Cell regulatory genes, encoded products, and uses related thereto

ABSTRACT

This application describes the cloning of p63, a gene at chromosome 3 q 27-29, that bears homology to the tumor suppressor p53. The p63 gene encodes at least six different isotypes. p63 was detected in a variety of human and mouse tissue and demonstrates remarkably divergent activities, such as the ability to transactivate p53 reporter genes and induce apoptosis. Isotopes of p63 lacking a transactivation domain act as dominant negatives towards the transactivation by p53 and p63.

1. BACKGROUND OF THE INVENTION

[0001] Neoplasia is characterized by deregulated cell growth anddivision. Inevitably, molecular pathways controlling cell growth mustinteract with those regulating cell division. It was not until veryrecently, however, that experimental evidence became available to bringsuch connection to light. Cyclin A was found in association with theadenovirus oncoprotein E1A in virally transformed cells (Giordona et al.Cell 58:981 (1989); and Pines et al. Nature 346:760 (1990)). Thecell-cycle gene implicated most strongly in oncogenesis thus far is thehuman cyclin D1. It was originally isolated through geneticcomplementation of yeast G₁ cyclin deficient strains (Xiong et al. Cell65:691(1991); and Lew et al. Cell 66:1197 (1991)), as cellular geneswhose transcription is stimulated by CSF-1 in murine macrophages(Matsushine et al. Cell 65:701 (1991)) and in the putative oncogene PRAD1 rearranged in parathyroid tumors (Montokura et al. Nature 350:512(1991).

[0002] However, the creation of a mutant onocogene is only one of therequirements needed for tumor formation; tumorigenesis appears to alsorequire the additional inactivation of a second class of critical genes:the “anti-oncogenes” or “tumor-suppressing genes.” Tumor suppressorgenes are a family of genes that negatively regulate cell growth and arelost or inactivated in most cancers. In their natural state these genesact to suppress cell proliferation. Damage to such genes leads to a lossof this suppression, and thereby results in tumorigenesis. Thus, thederegulation of cell growth may be mediated by either the activation ofoncogenes or the inactivation of tumor-suppressing genes (Weinberg, R.A., (Sept 1988) Scientific Amer.pp 44-51).

[0003] Oncogenes and tumor-suppressing genes have a basic distinguishingfeature. The oncogenes identified thus far have arisen only in somaticcells, and thus have been incapable of transmitting their effects to thegerm line of the host animal. In contrast, mutations intumor-suppressing genes can be identified in germ line cells, and arethus transmissible to an animal's progeny.

[0004] The classic example of a hereditary cancer is retinoblastomas inchildren. The incidence of the retinoblastomas is determined by a tumorsuppressor gene, the retinoblastoma (RB) gene (Weinberg, R. A., (Sept1988) Scientific Amer. pp 44-51; Hansen et al. (19881 Trends Genet4:125-128). Individuals born with a lesion in one of the RB alleles arepredisposed to early childhood development of retinoblastomas.Inactivation or mutation of the second RB allele in one of the somaticcells of these susceptible individuals appears to be the molecular eventthat leads to tumor formation (Caveneee et al. (1983) Nature305:799-784; Friend et al. (1987) PNAS 84:9059-9063).

[0005] The RB tumor-suppressing gene has been localized onto humanchromosome 13. The mutation may be readily transmitted through the germline of afflicted individuals (Cavenee, et al. (1986) New Engl. J. Med314:1201-1207). Individuals who have mutations in only one of the twonaturally present alleles of this tumor-suppressing gene are predisposedto retinoblastoma. Inactivation of the second of the two alleles is,however, required for tumorigenesis (Knudson (1971) PNAS 68:820-823).

[0006] A second tumor-suppressing gene is the p53 gene (Green (1989)Cell 56:1-3; Mowat et al (1985 Nature 314:633-636). The protein encodedby the p53 gene is a nuclear protein that forms a stable complex withboth the SV40 large T antigen and the adenovirus E1B 55 kd protein. Thep53 gene product may be inactivated by binding to these proteins.

[0007] Based on cause and effect analysis of p53 mutants, the functionalrole of p53 as a “cell-cycle checkpoint”, particularly with respect tocontrolling progression of a cell from G1 phase into S phase, hasimplicated p53 as able to directly or indirectly affect cycle cylemachinery. The first firm evidence for a specific biochemical linkbetween p53 and the cell-cycle comes a finding that p53 apparentlyregulates expression of a second protein, p21, which inhibitscyclin-dependent kinases (CDKs) needed to drive cells through thecell-cycle, e.g. from G1 into S phase. For example, it has beendemonstrated that non-viral transformation, such as resulting at leastin part from a mutation of deletion of of the p53 tumor suppressor, canresult in loss of p21 from cyclin/CDK complexes. As described by Xionget al. (1993) Nature 366:701-704, induction of p21 in response to p53represents a plausible mechanism for effecting cell-cycle arrest inresponse to DNA damage, and loss of p53 may deregulate growth by loss ofthe p21 cell-cycle inhibitor.

[0008] More recently, researchers discovered yet another tumorsuppressing gene, p73, which closely resembles p53. Not only does thisprotein bear a strong structural identity with p53, it also possesssimilar functional attributes. For instance, this protein disclosed thegrowth-inhibiting and apoptosis promoting effects, it triggered p21production, suggesting thereby that it inhibited cell growth through thesame pathway as that used by p53. Here, we describe the discovery of anovel family of cell regulatory genes, the p-63 family, which exhibitsconsiderable sequence identity with p53 and and p73, and appears topossess similar functional attributes.

2. SUMMARY OF THE INVENTION

[0009] The p53 tumor suppressor protein is involved in multiple centralcellular processes, including transcription, DNA repair, genomicstability, senescence, cell cycle control and apoptosis. p53 isfunctionally inactivated by structural mutations, interaction with viralproducts, and endogenous cellular mechanisms in the majority of humancancers. In fact, the p53 protein is one of the most frequently mutatedtumor suppressor to be identified in human cancers. More than 50% ofprimary human tumor cells over-express a variety of mutant p53 forms.p73 which shares considerable structural identity maps to chromosomalregion I p36, a region which is frequently deleted in neuroblastoma andother tumors.

[0010] Here we describe a third family of cell regulatory genes,encoding the p63-family of proteins which also demonstrate considerablestructural or sequence identity to the DNA-binding, oligomerization, andtransactivation domains of p53. p63 differs from p53 in that multiplep63 transcripts yielding six major protein products have been discoveredby cDNA cloning. For example, the six major p63 products are listed inFIG. 2C. It was found that unlike p53, the p63 gene encodes multipleisotypes with remarkably divergent abilities. For instance, p63 variantspossessing the N-terminus, i.e., TAp63γ, showed strong transactivationand cell-death inducing abilities. TAp63γ transactivates p53 reportergenes and may induce apoptosis. In addition, it was found that thepredominant p63 isotypes in many epithelial tissues lack an acidicN-terminus corresponding to the transactivation domain of p53. p63variants which lack the transactivation domain, i.e., ΔNp63α, ΔNp63γ,suppressed transactivation by both p53 and p63. Additionally, thesevariants lacking the N-terminus may possibly regulate growth and mayplay an essential role in the regenerative processes, particularlyregeneration of epithelial tissue. In one aspect, the inventiondiscloses that these truncated p63 variants can act as dominant-negativeagents towards transactivation by p53, thereby suggesting thepossibility of physiological interactions amongst members of the p53family. Examples of these variants include the disclosed ΔNp63α andΔNp63γ. Thus, in one embodiment, the p63 family of cell-regulatoryproteins are involved in the modulation of cell growth or regulate thegrowth phenotype of a cell.

[0011] One aspect of the invention features a substantially purepreparation of a cell regulatory protein, or a fragment thereof, thefull-length form of the cell regulatory protein having an amino acidsequence at least 70% homologous to the amino acid sequence representedin one of SEQ ID Nos. 13-24; the polypeptide has an amino acid sequenceat least 80% homologous to the amino acid sequence represented in one ofSEQ ID Nos. 13-24; the polypeptide has an amino acid sequence at least90% homologous to the amino acid sequence represented in one of SEQ IDNos. 13-24; the polypeptide has an amino acid sequence at least 95%homologous to the amino acid sequence represented in one of SEQ ID Nos.13-24; the polypeptide has an amino acid sequence identical to the aminoacid sequence represented in one of SEQ ID Nos. 13-24. In a preferredembodiment: the fragment comprises at least 5 contiguous amino acidresidues of SEQ ID Nos. 13-24; the fragment comprises at least 20contiguous amino acid residues of SEQ ID Nos. 13-24; the fragmentcomprises at least 50 contiguous amino acid residues of SEQ ID Nos.13-24.

[0012] In yet another embodiment, the fragment includes the DNA bindingdomain of p63 and comprises at least 5 contiguous amino acid residues ofSEQ ID Nos. 13-24; the fragment includes the DNA binding domain of p63and comprises at least 20 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the DNA binding domain of p63 and comprisesat least 50 contiguous amino acid residues of SEQ ID Nos. 13-24.

[0013] The DNA binding domain of these nucleic acid transcripts areremarkably conserved. The six variants demonstrate 100% identity in thisregion and this domain also demonstrates considerable identity to thecorresponding DNA-binding domains of p53 and p73. It was found that p63variants bound the target sequences which are bound by p53, for example,ΔNp63γ, p53, and Ta p63α all yielded significant mobility shifts withthree separate oligonucleotides, specifically, with a minimal p53binding sequence, a p53 binding site in the p21 promoter WAF, and amutant p53 binding site. Results of the assay are shown in FIG. 25.

[0014] Another aspect of the present invention features a polypeptide,of the cell regulator protein family, which functions in one of eitherrole of an agonist of cell-cycle regulation or an antagonist ofcell-cycle regulation. In a preferred embodiment: the subject cellregulator-protein specifically binds a target DNA or protein; e.g.specifically binds a target DNA; e.g. is reasonably expected totransactivate genes involved in cell cycle arrest, such as p21;interacts with the DNA repair and synthetic machinery, such asproliferating cellular nuclear antigen, GADD 45, or proteins modulatingapoptosis. In a more preferred embodiment, the cell regulator-proteinregulates aand/or modulates growth of an eukaryotic cell-cycle, e.g. amammalian cell-cycle, e.g., a human cell-cycle; the cell regulatorprotein inhibits cell growth of a eukaryotic cell, e.g., a human cell;the tumor suppressor-protein inhibits progression of a eukaryotic cellfrom G1 phase into S phase, e.g., inhibits progression of a mammaliancell from G1 phase into S phase, e.g., inhibits progression of a humancell from G1 phase into S phase; the cell regulator-protein suppressestumor growth, e.g. in a tumor cell, e.g. in a tumor cell having anunimpaired p53 or p63 or p53-like protein checkpoint. Yet another aspectof the present invention concerns an immunogen comprising a cellregulator-protein of the present invention, or a fragment thereof, in animmunogenic preparation, the immunogen being capable of eliciting animmune response specific for the cell regulator-protein; e.g. a humoralresponse, e.g., an antibody response; e.g. a cellular response. Thus, inone embodiment, the p-63 family of cell-regulatory proteins are involvedin the modulation of cell growth or regulate the growth phenotype of acell.

[0015] Another aspect of the present invention features recombinant cellregulator-protein, or a fragment thereof, cell regulatory protein, or afragment thereof, the full-length form of the cell regulatory genesprotein having an amino acid sequence at least 70% homologous to theamino acid sequence represented in one of SEQ ID Nos. 13-24; thepolypeptide has an amino acid sequence at least 80% homologous to theamino acid sequence represented in one of SEQ ID Nos. 13-24; thepolypeptide has an amino acid sequence at least 90% homologous to theamino acid sequence represented in one of SEQ ID Nos. 13-24; thepolypeptide has an amino acid sequence at least 95% homologous to theamino acid sequence represented in one of SEQ ID Nos. 13-24; thepolypeptide has an amino acid sequence identical to the amino acidsequence represented in one of SEQ ID Nos. 13-24. In a preferredembodiment: the fragment comprises at least 5 contiguous amino acidresidues of SEQ ID Nos. 13-24; the fragment comprises at least 20contiguous amino acid residues of SEQ ID Nos. 13-24; the fragmentcomprises at least 50 contiguous amino acid residues of SEQ ID Nos.13-24.

[0016] In yet another embodiment, the fragment includes the DNA bindingdomain of p63 and comprises at least 5 contiguous amino acid residues ofSEQ ID Nos. 13-24; the fragment includes the DNA binding domain of p63and comprises at least 20 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the DNA binding domain of p63 and comprisesat least 50 contiguous amino acid residues of SEQ ID Nos. 13-24.

[0017] In yet another embodiment, the fragment includes the core domainof p63 and comprises at least 5 contiguous amino acid residues of SEQ IDNos. 13-24; the fragment includes the core domain of p63 and comprisesat least 20 contiguous amino acid residues of SEQ ID Nos. 13-24; thefragment includes the core domain of p63 and comprises at least 50contiguous amino acid residues of SEQ ID Nos. 13-24. The core domainextends from about amino acids “PQV” through about amino acid “HLLQ”,for instance, in TAp63γ, this region extends from about amino acid 70through about amino acid 409.

[0018] In a preferred embodiment, the recombinant cell regulator-proteinfunctions in one of either role of an agonist of cell-cycle regulationor an antagonist of cell-cycle regulation. In a preferred embodiment:the subject p63 protein specifically binds a target DNA or protein; e.g.specifically binds a target DNA; e.g. is reasonably expected totransactivate genes involved in cell cycle arrest, such as p21; interactwith the DNA repair and synthetic machinery, such as proliferatingcellular nuclear antigen, GADD 45, or proteins modulating apoptosis. Ina more preferred embodiment: the p63 protein regulates and/or modulatesgrowth of an eukaryotic cell-cycle, e.g. a mammalian cell-cycle, e.g., ahuman cell-cycle; the p63 protein inhibits cell growth of a eukaryoticcell, e.g., a human cell; the p63 protein inhibits progression of aeukaryotic cell from G1 phase into S phase, e.g., inhibits progressionof a mammalian cell from G1 phase into S phase, e.g., inhibitsprogression of a human cell from G1 phase into S phase; the tumorsuppressor-protein suppresses tumor growth, e.g. in a tumor cell, e.g.in a tumor cell having an unimpaired p53 or p63 or p53-like proteincheckpoint. Thus, in one embodiment, the p-63 family of cell-regulatoryproteins are involved in the modulation of cell growth or regulate thegrowth phenotype of a cell.

[0019] In yet other preferred embodiments, the recombinanttumor-suppressor-protein is a fusion protein further comprising a secondpolypeptide portion having an amino acid sequence from a proteinunrelated the protein of SEQ ID Nos. 13-24. Such fusion proteins can befunctional in a two-hybrid assay.

[0020] Another aspect of the present invention provides a substantiallypure nucleic acid having a nucleotide sequence which encodes a cellregulator-protein, or a fragment thereof, having an amino acid sequenceat least 70% homologous to one of SEQ ID Nos. 13-24. In a more preferredembodiment: the nucleic acid encodes a protein having an amino acidsequence at least 80% homologous to SEQ ID No. 5, more preferably atleast 90% homologous to SEQ ID No. 5, and most preferably at least 95%homologous to SEQ ID No. 5; the nucleic acid encodes a protein having anamino acid sequence at least. 80% homologous to SEQ ID No. 8, morepreferably at least 90% homologous to SEQ ID No. 8, and most preferablyat least 95% homologous to SEQ ID No. 8 the nucleic acid encodes aprotein having an amino acid sequence at least 80% homologous to SEQ IDNo. 7, more preferably at least 90% homologous to SEQ ID No. 7, and mostpreferably at least 95% homologous to SEQ ID No. 7.

[0021] In a preferred embodiment: the subject cell regulator-proteinspecifically binds a target DNA or protein; for example transactivategenes involved in cell cycle arrest, such as p21; interact with the DNArepair and synthetic machinery, such as proliferating cellular nuclearantigen, GADD 45, or proteins modulating apoptosis. In a more preferredembodiment: the cell regulator-protein regulates and/or modulates growthof an eukaryotic cell-cycle, e.g. a mammalian cell-cycle, e.g., a humancell-cycle; the cell regulator-protein inhibits cell growth of aeukaryotic cell, e.g., a human cell; the cell regulator-protein inhibitsprogression of a eukaryotic cell from G1 phase into S phase, e.g.,inhibits progression of a mammalian cell from G1 phase into S phase,e.g., inhibits progression of a human cell from G1 phase into S phase;the cell regulator-protein suppresses tumor growth, e.g. in a tumorcell, e.g. in a tumor cell having an unimpaired p53 or p63 or p53-likeprotein checkpoint. Thus, in one embodiment, the p-63 family ofcell-regulatory proteins are involved in the modulation of cell growthor regulate the growth phenotype of a cell.

[0022] In another embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides of SEQ ID No. 1; more preferably to at least20 consecutive nucleotides of SEQ ID No. 1; more preferably to at least40 consecutive nucleotides of SEQ ID No. 1.

[0023] In a further embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides of SEQ ID No. 2; more preferably to at least20 consecutive nucleotides of SEQ ID No. 2; more preferably to at least40 consecutive nucleotides of SEQ ID No. 2.

[0024] In yet a further embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides of SEQ ID No. 3; more preferably to at least20 consecutive nucleotides of SEQ ID No. 3; more preferably to at least40 consecutive nucleotides of SEQ ID No. 3.

[0025] In yet a further embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides of SEQ ID No. 6; more preferably to at least20 consecutive nucleotides of SEQ ID No. 6; more preferably to at least40 consecutive nucleotides of SEQ ID No. 6.

[0026] Furthermore, in certain embodiments, the cell regulator nucleicacid will comprise a transcriptional regulatory sequence, e.g. at leastone of a transcriptional promoter or transcriptional enhancer sequence,operably linked to the cell regulator-gene sequence so as to render therecombinant cell regulator gene sequence suitable for use as anexpression vector.

[0027] The present invention also features transgenic non-human animals,e.g. mice, which either express a heterologous cell regulator-gene, e.g.derived from humans, or which mis-express their own cell regulator-gene,e.g. where p63, p53 or p73 expression is disrupted. Such a transgenicanimal can serve as an animal model for studying cellular disorderscomprising mutated or mis-expressed cell regulator allelles.

[0028] The present invention also provides a probe/primer comprising asubstantially purified oligonucleotide, wherein the oligonucleotidecomprises a region of nucleotide sequence which hybridizes understringent conditions to at least 10 consecutive nucleotides of sense orantisense sequence of one of SEQ ID Nos. 1-12, or naturally occurringmutants thereof. In preferred embodiments, the probe/primer furthercomprises a label group attached thereto and able to be detected, e.g.the label group is selected from a group consisting of radioisotopes,fluorescent compounds, enzymes, and enzyme co-factors. Such probes canbe used as a part of a diagnostic test kit for identifying transformedcells, such as for measuring a level of a p63, p73 or p53 encodingnucleic acid in a sample of cells isolated from a patient; e.g. formeasuring the mRNA level in a cell or determining whether the genomiccell regulator gene has been mutated or deleted.

[0029] The present invention also provides a method for treating ananimal having unwanted cell growth characterized by a loss of wild-typecell regulator-protein function, comprising administering atherapeutically effective amount of an agent able to transactivate genesinvolved in cell cycle arrest. For instance, a therapeutically effectiveamount of a p63 variant comprising a transactivating domain, for exampleTap63γ. In one embodiment, the method comprises administering a nucleicacid construct encoding a cell regulator protein, e.g. a polypeptiderepresented in one of SEQ ID Nos. 13-24, under conditions wherein theconstruct is incorporated by cell regulator-deficient cells and thepolypeptide is expressed, e.g. by gene therapy techniques. In anotherembodiment, the method comprises administering a cell regulator mimetic,e.g. a peptidomimetic, which binds to and transactivates genes involvedin cell-cycle arrest.

[0030] Another aspect of the present invention provides a method ofdetermining if a subject, e.g. a human patient, is at risk for adisorder characterized by unwanted cell proliferation, comprisingdetecting, in a tissue of the subject, the presence or absence of agenetic lesion characterized by at least one of (i) a mutation of a geneencoding a protein represented by one of SEQ ID Nos. 13-24, or a homologthereof; or (ii) the mis-expression of the cell regulator-gene, e.g. thep63, p53 or p73 gene. In preferred embodiments: detecting the geneticlesion comprises ascertaining the existence of at least one of adeletion of one or more nucleotides from said gene, an addition of oneor more nucleotides to said gene, an substitution of one or morenucleotides of said gene, a gross chromosomal rearrangement of saidgene, a gross alteration in the level of a messenger RNA transcript ofsaid gene, the presence of a non-wild type splicing pattern of amessenger RNA transcript of said gene, or a non-wild type level of saidprotein. For example, detecting the genetic lesion can comprise (i)providing a probe/primer comprising an oligonucleotide containing aregion of nucleotide sequence which hybridizes to a sense or antisensesequence of one of SEQ ID Nos. 1-12, or naturally occurring mutantsthereof, or 5′ or 3′ flanking sequences naturally associated with thecell regulator-gene; (ii) exposing the probe/primer to nucleic acid ofthe tissue; and (iii) detecting, by hybridization of the probe/primer tothe nucleic acid, the presence or absence of the genetic lesion; e.g.wherein detecting the lesion comprises utilizing the probe/primer todetermine the nucleotide sequence of the cell regulator-gene and,optionally, of the flanking nucleic acid sequences; e.g. whereindetecting the lesion comprises utilizing the probe/primer in apolymerase chain reaction (PCR); e.g. wherein detecting the lesioncomprises utilizing the probe/primer in a ligation chain reaction (LCR).In alternate embodiments, the level of said protein is detected in animmunoassay.

[0031] Yet another aspect of the invention pertains to a peptidomimeticwhich transactivates genes involved in cell-cycle arrest.

[0032] Like p53, p63 is believed to be a multifunctional protein thatexerts a variety of effects and plays a central role in the regulationof the cell cycle. For instance, over-expression of p63, particularlyp63 variants comprising a transactivating domain may induce growtharrest, associated with the G₀/G₁ checkpoint, apoptosis occurring eitherthrough the G₀G₁ checkpoint, or the S-phase or cell differentiation. Inparticular, p63 is implicated in the mechanism that senses damaged DNA,and controls its repair and in the

[0033] induction of cell death. It is possible that, this may beaccomplished by transactivation of the proliferating cell nuclearantigen (PCNA), involved in DNA replication and repair and the GADD45gene, whose product interacts with DNA. Furthermore, p63 may also bindseveral transcription associated proteins, which are involved in DNAdamage and repair machinery. The role of p63 in tumorigenesis may bedemonstrated by using p63 knocked out mice. Development of a highfrequency of tumors in adult life would be indicative that p63, likep53, functions as a cell regulator gene. It is known in the art that p53transactivates the pro-apoptotic gene, Bax (Miyashita and Reed 1995), inaddition to an array of genes responsive to oxidative stress. Based onthese observations p53 has been implicated in inducing cell-cycle arrestto allow for repair processes and/or the induction of cell death in theevent of unmitigated stress. It was seen that p63 variants comprising atransactivation domain, i.e., TAp63γ, exhibited strong transcriptionalactivation of the p53 reporter. p63 variants lacking thetransactivational domain, e.g., Δp63 are implicated in the regenerationof epithelial cells allowing proliferation of the epithelial cells. Itis interesting that the ΔN variants, or dominant negative, versions ofp63 seem to be the isotype expressed in many cancers. This may be tiedto the observation that many types of cancer, particularly cervicalcarcinoma, show an overexpression of chromosome 3q, where the p63 geneis located. If this chromosomal amplification results in theoverexpression of a ΔNp63 product which opposes p53 or transactivatingp63 forms, the experiments that follow show that the dominant negativevariants do in fact suppress the transactivating forms p63 and p53.Therefore, these p63 variants may be implicated in cancer biology andpossible diagnostic/prognostic applications.

[0034] Thus, in one embodiment, the p-63 family of cell-regulatoryproteins are involved in the modulation of cell growth or regulate thegrowth phenotype of a cell. Because of its various roles activation ofp63 may result in different outcomes as shown below:

[0035] Another aspect of the invention features related DNA andpolypeptide sequences which are characterized by a particular percenthomology or identity as determined by any of various mathematicalalgorithms known in the art. A number of mathematical algorithms havebeen developed to find and measure homology between two DNA orpolypeptide sequences. For example the local homology algorithm of Smithand Waterman ((1981)Advances in Applied Mathematics 2:482-89) is used inthe alignment software program called “BestFit,” which is available fromthe GCG software package (Genetics Computer Group, 575 Science Drive,Madison, Wis. 53711; www.gcg.com). Other methods for aligning sequencesinclude the homology alignment algorithm of Needleman and Wunsch ((1970)J. Mol. Biol. 48: 443) and the similarity search method of Pearson andLipman ((1988) Proc. Natl. Acad. Sci. (USA) 85: 2444. These algorithmsare available as computerized software such as GAP, FASTA and TFASTA inthe Wisconsin Genetic Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.). Alternatively, sequences can bealigned manually by inspection, and the best analysis, yielding thegreatest degree of homology, is chosen.

[0036] These methods to describe the sequence relationships between twoor more polynucleotides require the analysis of certain elements ofthese sequences and the defining of certain parameters with which toanalyze them. First, a “reference sequence” is a defined sequence usedas a basis for a sequence comparison. A reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence is given in a sequence listing or may comprise acomplete cDNA or gene sequence. Generally, a reference sequence is atleast 25 nucleotides in length, frequently at least 25 nucleotides inlength and often at least 50 nucleotides in length. Since twopolynucleotides may each comprise both a sequence that is similarbetween the two polynucleotides and a sequence that is divergent betweenthe two polynucleotides, sequence comparisons between two or morepolynucleotides typically performed by comparing sequences of the twopolynucleotides over a “comparison window” to identify and compare localregions of sequence similarity. A comparison window, as used herein,refers to a conceptual segment of at least 20 contiguous nucleotidepositions wherein a polynucleotide sequence may be compared to areference sequence of at least 20 contiguous nucleotides and wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e. gaps) of 20 percent or less ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences.

[0037] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims. Thepractice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

3. BRIEF DESCRIPTION OF THE FIGURES

[0038]FIG. 1. Primary Structure Alignments of p53, p73, and p63.

[0039] Human p53, human p73%, human Tap63 γ are presented, with residuesidentical to p53 shaded in gray, and remaining consensus residues shadedin black.

[0040]FIG. 2. Genomic Origin and Diversity of p63 Isotypes

[0041] (A) Schematic of human p63 gene structure highlighting positionsof exons (coding sequences in black), the two promoters in exon one(black arrow) and exon 3′ (gray arrow), and the majorpost-transcriptional splicing events which give rise to the major p63isotypes.

[0042] (B) Domain structure of p53, p73α and β, and the major p63isotypes, Tap63αβ, and γ, and ΔNp63 a, β, and γ, highlighting regionsinvolved in transactivation (TA), DNA binding, and oligomerization(oligo). White box denotes 39aa N-terminal extension unique to TA*p63.Gray box represents 14aa unique to ΔNp63.

[0043] (C) Sequence alignment of N-termini of murine and human p63including that found in TA*p63, TAp63, and ΔNp63.

[0044] (D) Alignment and comparison of the human p63 a, β, and γC-terminal sequences.

[0045]FIG. 3. Chromosomal Localization of Human and Mouse p63 Gene.

[0046] (A) Schematic of chromosome 3 showing localization of human p63gene at 3q27-29 based on fluorescence in situ hybridization with a p63genomic PAC clone.

[0047] (B) Schematic of proximal end of mouse chromosome 16 showinglocation of murine p63 gene, as determined by linkage analysis againstJackson Laboratory interspecific backcross panels BBS and BSB. Locimapping to similar positions are presented in alphabetical order, andmissing typing inferred from surrounding data where assignment wasunambiguous.

[0048]FIG. 4. Immunolocalization of p63 in Human Epithelial Tissues.

[0049] Paraffin sections of normal human epithelial tissues probed withmonoclonal antibodies to p63 using an alkaline phosphatase reportersystem.

[0050] (A) p63 staining in foreskin showing nuclear localization of p63in basal epithelial cells.

[0051] (B) p63 localization to basal cells of ectocervical epithelium.

[0052] (C) p63 localization in basal cells of vaginal epithelium.

[0053] (D) p63 staining of basal cells of urothelium.

[0054] (E) p63 staining of epithelial cell layer below luminal cells inprostate.

[0055]FIG. 5. Tissue Distribution of p63 Isotypes

[0056] (A) RT-PCR analysis of total RNA prepared from various adultmouse tissues using oligonucleotide primers designed to amplify TAp63isotypes, revealing a ˜410pb product in heart, testis, kidney/adrenal,thymus, brain, and cerebellum.

[0057] (B) RT-PCR analysis using template RNA in (A) witholigonucleotide primers designed to yield a ˜240 bp product for ΔNp63isotypes, revealing expected product in kidney/adrenal, spleen, andthymus.

[0058] (C) Tel electrophoresis of RNA used as template in RT-PCRanalyses to determine template integrity.

[0059] (D) Analysis of p63 transcripts in human epithelial tissues.RT-PCR analyses of RNA from primary human foreskin keratinocytes,ectocervical cells, and the human cervical carcinoma cell line ME180using oligonucleotides designed to amplify TAp63 transcripts (leftpanel) and ΔNp63 transcripts (right panel). The ME180 cells showproducts corresponding to both the TAp63 and the ΔNp63 transcripts,while RNA from primary keratinocytes and ectocervical cells yieldpredominantly products from ΔNp63 transcripts.

[0060] (E) Western blot of primary human foreskin keratinocytes (1°HFK), ME180 human cervical carcinoma cells (ME180), and BHK cellsexpressing epitope-tagged p63 isotypes (TA*p63γ, TAp63γ, ΔNp63γ,TA*p63a, TAp63a, and ΔNp63a) using the 4A4 anti-p63 monoclonal antibody.The major p63 species in primary keratinocytes migrates slightly fasterthan the epitopetagged ΔNp63a protein.

[0061]FIG. 6. Transactivation of p53-Reporter Genes by p63 IsotypesTranscriptional activation of p53-reporter gene in Saos-2 cellstransfected with the indicated p53 and p63 expression constructs.Chemiluminescence signal from reporter P-galactosidase assays wereperformed and normalized for transfection efficiency using assays forco-transfected, constitutively expressed luciferase vectors. Error barsindicate standard deviation in triplicate assays.

[0062]FIG. 7. Induction of Apoptosis by p63 Isotypes

[0063] BHK cells transfected with idential amounts of wildtype p53 (A)mutant p53 (B), TAp63γ (C), ΔNp63γ(D), and ΔNp63a(E), were processed forimmunofluorescence after 16 hours using epitope-tagged antibodies (leftpanel) and Hoechst dye for DNA staining (right panel). Wildtype p53- andTAp63γ-expressing cells showed high levels of apoptosis (arrows) despitevery low protein expression, while ΔNp63γ yielded high proteinexpression and modest levels of apoptosis. Mutant p53 and ΔNp63a showedhigh levels of protein expression but control levels of apoptosis.

[0064]FIG. 8. Interactions Amongst p63 Isotypes and p53 inTransactivation Assays

[0065] (A) Transactivation analysis in Saos-2 cells transfected with aconstant amount of wild-type p53 expression vector, minimal p53-reporterconstruct, and either ΔNp63γ or ΔNp63a expression vectors at ratios of1:5 or 1:1 with respect to p53, as indicated.

[0066] (B) Transactivation analysis in Saos-2 cells transfected with aconstant amount of TAp63γ expression vector, p53-reporter construct, andeither ΔNp63γ or ΔNp63a expression vectors at ratios of 1:5 or 1:1 withrespect to TAp63γ, as indicated.

[0067]FIG. 9. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 1 and 13 respectively.

[0068]FIG. 10. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 2 and 14 respectively.

[0069]FIG. 11. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 3 and 15 respectively.

[0070]FIG. 12. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 4 and 16 respectively.

[0071]FIG. 13. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 5 and 17 respectively.

[0072]FIG. 14. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 6 and 18 respectively.

[0073]FIG. 15. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 7 and 19 respectively.

[0074]FIG. 16. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 8 and 20 respectively.

[0075]FIG. 17. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 9 and 21 respectively.

[0076]FIG. 18. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 10 and 22 respectively.

[0077]FIG. 19. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 11 and 23 respectively.

[0078]FIG. 20. Represents nucleic acid and amino acid sequencesrepresented by SEQ ID Nos.: 12 and 24 respectively.

[0079]FIG. 21 shows induction of transactivating version of p63 upon UVirradiation. The time course is similar, but not identical, to p53'sinduction by UV. This is the first demonstration that p63 (and inparticular, the transactivating, p53-like, version) can respond tostress signals such as UV/DNA damage.

[0080]FIG. 22 shows that p63 protein levels, while high in the basal,proliferative/regenerative layer of squamous epithelia, decreasesdramatically upon differentiation/maturation of these keratinocytes.This may implicate p63 in differntiation processes that are importantfor both oncogenesis and normal development.

[0081]FIG. 23 shows p63 RNA expression in some human cancer cell lines,mostly cervical carcinoma.

[0082]FIG. 24 just shows p63 RNA expression in some human breast cancercell lines. It is useful to note that p63 is expressed in these cancers.ΔN versions also strongly expresses in some of these lines.

[0083]FIG. 25. Shows the results of an electrophoretic shift assay.

4. DETAILED DESCRIPTION OF THE INVENTION

[0084] 4.1. General

[0085] The present invention concerns the discovery of a new family ofcell regulatory proteins, referred to herein as the p63 family ofproteins, which demonstrate certain sequence identity to known tumorsuppressor proteins p53 and p73. The p63 proteins may generally berepresented by the general formula: X-Y-Z, wherein X represents theN-termini of the proteins, e.g. a ΔN, a TA*, or TA polypeptide sequence(infra), Y represents the core domain of the protein, and Z representsthe C termini, e.g., the α, β, or γ polypeptide sequences (infra). Themouse and human p63 were identified by using a novel PCR based strategy.Specifically, it was observed that the intron-exon organization wasconserved between p53 and p73, by using the known exon and intron sizesfor these genes it was possible to amplify portions of two adjacentexons and the intervening intron. The rationale being that sequencesimilarities between the exonic regions would demonstrate a relatedgene, while differences in size would indicate a novel family member. Bythis technique we identified at least one new paralog of the p53/p73/p63related family. Mouse cDNA was isolated using the RACE (5′ rapidamplification of cDNA ends) technique and the sequencing of theamplification product indicated that the amplified cDNA possessed atruncated N-terminus, i.e. the transactivation domain was absent in thisproduct. Additional splice variants of the mouse p63 were identified byscreening a cDNA library with a probe corresponding to exons 5 through 9of p63. In general, splice variants differing in the C-terminus havebeen designated as α, β, and γ forms, whereas p63 members differing inthe N-terminus are designated as the ΔN and TA forms, wherein the ΔNform lack the transactivational domain.

[0086] The appended sequence listing, provides a list of the nucleicacid and protein sequences that are included within the scope of thisinvention. TABLE 1 Guide to p63 sequences in Sequence Listing NucleotideSequence Amino Acid Sequence hu-TAp63α SEQ ID No. 1 SEQ ID No. 13hu-TAp63β SEQ ID No. 2 SEQ ID No. 14 hu-TAp63γ SEQ ID NO. 3 SEQ ID NO.15 hu-ΔNp63α SEQ ID No. 4 SEQ ID No. 16 hu-ΔNp63β SEQ ID No. 5 SEQ IDNo. 17 hu-ΔNp63γ SEQ ID No. 6 SEQ ID No. 18 mu-TA*p63α SEQ ID No. 7 SEQID No. 19 mu-TA*p63β SEQ ID No. 8 SEQ ID No. 20 mu-TA*p63γ SEQ ID No. 9SEQ ID No. 21 mu-ΔNp63α SEQ ID No. 10 SEQ ID No. 22 mu-ΔNp63β SEQ ID No.11 SEQ ID No. 23 mu-ΔNp63γ SEQ ID No. 12 SEQ ID No. 24

[0087] By fluorescence in situ hybridization (FISH), the human p63 genehas been localized to chromosomal position 3q27-29. Early expressiondata suggests that p63 is expressed at steady-state detectable levels invarious adult tissues. The p63 proteins can be divided into twoclasses—one with p53-like properties and the other lackingp53-associated functions such as transcriptional activation andapoptosis. p63 transcripts were detected in a wide range of adulttissues, including heart, testis, kidney/adrenal, spleen, thymus, andbrain, typically showing a predominance of either the TA or AN isotypes.Analysis of human epithelial tissues has provided further insights intoendogenous p63 expression, as immunohistochemistry with anti-p63monoclonal antibodies revealed strong and discrete labeling of thenuclei of basal cells within the epidermis, ectocervical epithelium,urothelium, and prostate epithelium, while more differentiated,suprabasal cells showed little or no labeling. The presence of p63 inbasal cell layers of epithelial tissues is significant because thesecells have an essential role in the regenerative processes of theseepithelia. Specifically, the basal cells are thought to be progenitor,or stem, populations for suprabasal layers, and as such are theproliferative components of these tissues. This finding that theseepithelial cells predominantly express ΔNp63 isotypes is consistent withgrowth-permitting requirements of proliferating cells, and may underliethe regenerative abilities of normal epithelial tissues.

[0088] It is contemplated by the present invention that the clonedp63-genes set out in the appended sequence listing, in addition torepresenting a inter-species family of related genes, are also each partof an intra-species family. That is, it is anticipated that otherparalogs of the human and mouse p63 proteins exist in those animals, andorthologs of each p63 gene are conserved amongst other animals. Forinstance, at low to medium stringency conditions, another transcript wasobserved and this probably represents a new paralogous gene related tothe p/53/p73/p63 family of genes, or may a splice variant of p63 as setforth in SEQ ID No. 1.

[0089] The p53 protein consists of 393 amino acids with variousfunctional domains, evolutionarily conserved domains and regions whichhave been designated as mutational hotspots. The functional domainsinclude: a transactivational domain (amino acids 20-50), sequencespecific DNA binding domain (amino acids 100-293) nuclear localizationsequence (amino acids 316-325), and the oligomerization domain (aminoacids 319-360). It was found that the homology between p73 and p53 wasconsiderable within the most conserved p53 domains. Thetransactivational domain (amino acids 1-45) exhibited 29% identity, theDNA binding region 63% identity (amino acids 113-290) and theoligomerization domain 38% identity with p53 (amino acids 319-363).

[0090] Interestingly it was observed that the sequence identity betweenthe mouse and human p63 sequences was considerably high, both at thenucleotide and protein levels, specifically, the mouse and human p63beta forms exhibit 90.8% identity at the DNA level and 98.6% identity atthe protein level, the alpha forms showed 83.4% at the DNA level and97.8% at the protein level. The sequence identity between p63 and p73alpha form is about 57.4% identity and the p63 and p73 beta form showsabout 69.7% identity at the protein level. p63 alpha form and p53exhibit 43.8% identity at the protein level.

[0091] Accordingly, certain aspects of the present invention relate tonucleic acids encoding p63 polypeptides, the p63 polypeptides themselves(including various fragments), antibodies immunoreactive with p63proteins, and preparations of such compositions. Moreover, the presentinvention provides diagnostic and therapeutic assays and reagents fordetecting and treating disorders involving, for example, aberrantexpression (or loss) of p63.

[0092] In addition, drug discovery assays are provided for identifyingagents which can modulate the biological function of p63 proteins, suchas by altering the binding of p63 molecules to target proteins or DNAsequences, or other extracellular/matrix factors. Furthermore, based onthe considerable sequence identity with p53, the skilled artisan couldreasonably appreciate that the p63-family of proteins would play asignificant role as a cell regulator, a tumor suppressor, function incell cycle control various developmental processes, apoptosis, geneexpression and tumorigenesis. p63 may also be implicated inhematopoiesis, muscle wasting (e.g. cachexia) and neuronaldifferentiation (and degenerative disorders related thereto). It isknown that p53 probably exists as a tetramer and dominant negativemutants of p53, which function by overwhelming the wild-type protein andprevent it from functioning probably forms a heteromeric proteincontaining both the mutant and wild-type subunits in which the wild typesubunits are unable to function. In one aspect, the inventorsdemonstrate that the p63 protein products lacking the transactivationaldomains, for example the ΔNp63α and ΔNp63γ have dominant negativeeffects on the activity of p53. Similarly, these dominant negative formsmay exert their function by forming a heteromeric protein with eitherwild-type p53 or p63 and hence prevent the wild type protein fromfunctioning.

[0093] 4.2. Definitions

[0094] For convenience, the meaning of certain terms and phrases used inthe specification, examples, and appended claims, are provided below.

[0095] A “p63” cell regulatory protein as referred to herein, refers toproteins that may generally be represented by the formula: X-Y-Z,wherein X represents the N-termini of the proteins, e.g. a TA, TA*, orΔN polypeptide sequence, Y represents the core domain of the protein,and Z represents the C termini, e.g., the α, β, or γ polypeptidesequences. Illustrative examples include proteins represented by SEQ IDNos. 13-24, and homologs thereof

[0096] A “p53 protein” refers to the sequence designated by GenBankAccession Number K03199 and orthologs thereto.

[0097] “P73” refers to the sequences disclosed by Kaghad et al., Cell90:809-819 (1997).

[0098] The term “agonist”, as used herein, is meant to refer to an agentthat mimics or upregulates (e.g. potentiates or supplements) p63bioactivity. A p63 agonist can be a wild-type p63 protein or derivativethereof having at least one bioactivity of the wild-type p63. A p63agonist can also be a compound that upregulates expression of a gene orwhich increases at least one bioactivity of a p63 protein. An agonistcan also be a compound which increases the interaction of a p63polypeptide with another molecule, e.g, a target peptide or nucleicacid.

[0099] “Antagonist” as used herein is meant to refer to an agent thatdownregulates (e.g. suppresses or inhibits) at least one p63bioactivity. A p63 antagonist can be a compound which inhibits ordecreases the interaction between a p63 protein and another molecule,e.g., a target peptide, such as angiotensin I or a kinin. Accordingly, apreferred antagonist is a compound which inhibits or decreaseshydrolysis of a target peptide. An antagonist can also be a compoundthat downregulates expression of a p63 gene or which reduces the amountof p63 protein present.

[0100] The term “antibody” as used herein is intended to wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includesfragments thereof which are also specifically reactive with anvertebrate, e.g., mammalian, p63 protein. Antibodies can be fragmentedusing conventional techniques and the fragments screened for utility inthe same manner as described above for whole antibodies. Thus, the termincludes segments of proteolytically-cleaved or recombinantly-preparedportions of an antibody molecule that are capable of selectivelyreacting with a p63 protein. Nonlimiting examples of such proteolyticand/or recombinant fragments include Fab, F(ab′)2, Fab′, Fv, fragments,and single chain antibodies (scFv) containing a V[L] and/or V[H] domainjoined by a peptide linker. The scFv's may be covalently ornon-covalently linked to form antibodies having two or more bindingsites. The subject invention includes polyclonal, monoclonal, or otherpurified preparations of antibodies and recombinant antibodies.

[0101] The term “allele”, which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide, or several nucleotides, and caninclude substitutions, deletions, and/or insertions of nucleotides. Anallele of a gene can also be a form of a gene containing mutations.

[0102] The term “allelic variant of a polymorphic region of an p63 gene”refers to a region of the p63 gene having one of several nucleotidesequences found in that region of the gene in other individuals.

[0103] The phenomenon of “apoptosis” is well known, and can be describedas a programmed death of cells. As is known, apoptosis is contrastedwith “necrosis”, a phenomenon when cells die as a result of being killedby a toxic material, or other external effect. Apoptosis involveschromatic condensation, membrane blebbing, and fragmentation of DNA, allof which are generally visible upon microscopic examination.

[0104] “Biological activity” or “bioactivity” or “activity” or“biological function”, which are used interchangeably, for the purposesherein means an effector or antigenic function that is directly orindirectly performed by a p63 polypeptide (whether in its native ordenatured conformation), or by any subsequence thereof. Biologicalactivities include binding to polypeptides, particularly in theformation of homomeric complexes or heteromeric complexes with other p53or p73 homologs, binding to other proteins or molecules; activity as aDNA binding protein, as a transcription regulator, ability to binddamaged DNA etc. A p63 bioactivity can be modulated by directlyaffecting the p63 polypeptide. Alternatively, an p63 bioactivity can bealtered by modulating the level of the p63 polypeptide, such as bymodulating expression of the p63 gene.

[0105] As used herein the term “bioactive fragment of a p63 polypeptide”refers to a fragment of a full-length p63 polypeptide, wherein thefragment specifically agonizes (mimics) or antagonizes (inhibits) theactivity of a wild-type p63 polypeptide. The bioactive fragmentpreferably is a fragment capable of interacting with at least one othermolecule, protein or DNA, with which a full length p63 protein can bind.

[0106] The term “an aberrant activity”, as applied to an activity of apolypeptide such as p63, refers to an activity which differs from theactivity of the wild-type or native polypeptide or which differs fromthe activity of the polypeptide present in a healthy subject. Anactivity of a polypeptide can be aberrant because it is stronger thanthe activity of its native counterpart. Alternatively, an activity canbe aberrant because it is weaker or absent relative to the activity ofits native counterpart. An aberrant activity can also be a change in theactivity; for example, an aberrant polypeptide can interact with adifferent target peptide. A cell can have an aberrant p63 activity dueto overexpression or underexpression of the gene encoding p63.

[0107] “Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0108] A “chimeric polypeptide” or “fusion polypeptide” is a fusion of afirst amino acid sequence encoding one of the subject p63 polypeptideswith a second amino acid sequence defining a domain (e.g. polypeptideportion) foreign to and not substantially homologous with any domain ofa p63 polypeptide. A chimeric polypeptide may present a foreign domainwhich is found (albeit in a different polypeptide) in an organism whichalso expresses the first polypeptide, or it may be an “interspecies”,“intergenic”, etc. fusion of polypeptide structures expressed bydifferent kinds of organisms. In general, a fusion polypeptide can berepresented by the general formula (X)_(n)-(Y)_(m)-(Z)_(n), wherein Yrepresents a portion of the p63 polypeptide, and X and Z are eachindependently absent or represent amino acid sequences which are notrelated to the native p63 sequence found in an organism, or which arenot found as a polypeptide chain contigous with the p63 sequence, wherem is an integer greater than or equal to one, and each occurence of nis, indepenedently, 0 or an integer greater than or equal to 1 (n and mare preferably no greater than 5 or 10).

[0109] The term “nucleotide sequence complementary to the nucleotidesequence set forth in SEQ ID NO. x” refers to the nucleotide sequence ofthe complementary strand of a nucleic acid strand having SEQ ID NO. x.The term “complementary strand” is used herein interchangeably with theterm “complement”. The complement of a nucleic acid strand can be thecomplement of a coding strand or the complement of a non-coding strand.

[0110] A “delivery complex” shall mean a targeting means (e.g. amolecule that results in higher affinity binding of a gene, protein,polypeptide or peptide to a target cell surface and/or increasedcellular or nuclear uptake by a target cell). Examples of targetingmeans include: sterols (e.g. cholesterol), lipids (e.g. a cationiclipid, virosome or liposome), viruses (e.g. adenovirus, adeno-associatedvirus, and retrovirus) or target cell specific binding agents (e.g.ligands recognized by target cell specific receptors). Preferredcomplexes are sufficiently stable in vivo to prevent significantuncoupling prior to internalization by the target cell. However, thecomplex is cleavable under appropriate conditions within the cell sothat the gene, protein, polypeptide or peptide is released in afunctional form.

[0111] As is well known, genes or a particular polypeptide may exist insingle or multiple copies within the genome of an individual. Suchduplicate genes may be identical or may have certain modifications,including nucleotide substitutions, additions or deletions, which allstill code for polypeptides having substantially the same activity. Theterm “DNA sequence encoding a p63 polypeptide” may thus refer to one ormore genes within a particular individual. Moreover, certain differencesin nucleotide sequences may exist between individual organisms, whichare called alleles. Such allelic differences may or may not result indifferences in amino acid sequence of the encoded polypeptide yet stillencode a polypeptide with the same biological activity.

[0112] A disease, disorder or condition “associated with” or“characterized by” an aberrant p63 activity refers to a disease,disorder or condition in a subject which is caused by or contributed toby an aberrant p63 activity.

[0113] The term “equivalent” is understood to include nucleotidesequences encoding functionally equivalent p63 polypeptides orfunctionally equivalent peptides having an activity of an p63 proteinsuch as described herein. Equivalent nucleotide sequences will includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants; and will, therefore, includesequences that differ from the nucleotide sequence of the p63 gene shownin SEQ ID NOs: 1-12, due to the degeneracy of the genetic code.

[0114] As used herein, the terms “gene”, “recombinant gene” and “geneconstruct” refer to a nucleic acid comprising an open reading frameencoding a p63 polypeptide of the present invention, including both exonand (optionally) intron sequences.

[0115] A “recombinant gene” refers to nucleic acid encoding a p63polypeptide and comprising p63-encoding exon sequences, though it mayoptionally include intron sequences which are derived from, for example,a chromosomal p63 gene or from an unrelated chromosomal gene. Exemplaryrecombinant genes encoding the subject p63 polypeptide are representedin the appended Sequence Listing. The term “intron” refers to a DNAsequence present in a given p63-gene which is not translated intoprotein and is generally found between exons.

[0116] The term “growth” or “growth state” of a cell refers to theproliferative state of a cell as well as to its differentiative state.Accordingly, the term refers to the phase of the cell cycle in which thecell is, e.g., G0, G1, G2, prophase, metaphase, or telophase, as well asto its state of differentiation, e.g., undifferetiated, partiallydifferentiated, or fully differentiated. Without wanting to be limited,differentiation of a cell is usually accompanied by a decrease in theproliferative rate of a cell.

[0117] “Homology” or “identity” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules,with identity being a more strict comparison. Homology and identity caneach be determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. A degree of homology or similarity oridentity between nucleic acid sequences is a function of the number ofidentical or matching nucleotides at positions shared by the nucleicacid sequences. A degree of identity of amino acid sequences is afunction of the number of identical amino acids at positions shared bythe amino acid sequences. A degree of homology or similarity of aminoacid sequences is a function of the number of amino acids, i.e.structurally related, at positions shared by the amino acid sequences.An “unrelated” or “non-homologous” sequence shares less than 40%identity, though preferably less than 25% identity, with one of the p63sequences of the present invention.

[0118] The term “interact” as used herein is meant to include detectableinteractions (e.g. biochemical interactions) between molecules, such asinteraction between protein-protein, protein-nucleic acid, nucleicacid-nucleic acid, and protein-small molecule or nucleic acid-smallmolecule in nature.

[0119] The term “isolated” as used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject p63 polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks the p63gene in genomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Moreover, an “isolated nucleicacid” is meant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state. Theterm “isolated” is also used herein to refer to polypeptides which areisolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides.

[0120] The term “modulation” as used herein refers to both upregulation(i.e., activation or stimulation (e.g., by agonizing or potentiating))and downregulation (i.e. inhibition or suppression (e.g., byantagonizing, decreasing or inhibiting)).

[0121] The term “mutated gene” refers to an allelic form of a gene,which is capable of altering the phenotype of a subject having themutated gene relative to a subject which does not have the mutated gene.If a subject must be homozygous for this mutation to have an alteredphenotype, the mutation is said to be recessive. If one copy of themutated gene is sufficient to alter the genotype of the subject, themutation is said to be dominant. If a subject has one copy of themutated gene and has a phenotype that is intermediate between that of ahomozygous and that of a heterozygous subject (for that gene), themutation is said to be co-dominant.

[0122] The “non-human animals” of the invention include mammalians suchas rodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant p63 gene sis present and/or expressed or disrupted in sometissues but not others.

[0123] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

[0124] The term “polymorphism” refers to the coexistence of more thanone form of a gene or portion (e.g., allelic variant) thereof. A portionof a gene of which there are at least two different forms, i.e., twodifferent nucleotide sequences, is referred to as a “polymorphic regionof a gene”. A polymorphic region can be a single nucleotide, theidentity of which differs in different alleles. A polymorphic region canalso be several nucleotides long.

[0125] A “polymorphic gene” refers to a gene having at least onepolymorphic region.

[0126] As used herein, the term “promoter” means a DNA sequence thatregulates expression of a selected DNA sequence operably linked to thepromoter, and which effects expression of the selected DNA sequence incells. The term encompasses “tissue specific” promoters, i.e. promoters,which effect expression of the selected DNA sequence only in specificcells (e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

[0127] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

[0128] The term “recombinant protein” refers to a polypeptide of thepresent invention which is produced by recombinant DNA techniques,wherein generally, DNA encoding a p63 polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. Moreover, the phrase “derivedfrom”, with respect to a recombinant p63 gene, is meant to includewithin the meaning of “recombinant protein” those proteins having anamino acid sequence of a native p63 polypeptide, or an amino acidsequence similar thereto which is generated by mutations includingsubstitutions and deletions (including truncation) of a naturallyoccurring form of the polypeptide.

[0129] “Small molecule” as used herein, is meant to refer to acomposition, which has a molecular weight of less than about 5 kD andmost preferably less than about 4 kD. Small molecules can be nucleicacids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids orother organic (carbon containing) or inorganic molecules. Manypharmaceutical companies have extensive libraries of chemical and/orbiological mixtures, often fungal, bacterial, or algal extracts, whichcan be screened with any of the assays of the invention to identifycompounds that modulate a p63 bioactivity.

[0130] As used herein, the term “specifically hybridizes” or“specifically detects” refers to the ability of a nucleic acid moleculeof the invention to hybridize to at least approximately 6, 12, 15, 20,30, 50, 100, 150, 200, 300, 350, 400 or 425 contigous nucleotides of ap63 gene, such as designated in any one of SEQ ID Nos: 1-12, or asequence complementary thereto, or naturally occuring mutants thereof,such that it has less than 15%, preferably less than 10%, and morepreferably less than 5% background hybridization to a cellular nucleicacid (e.g. mRNA or genomic DNA) encoding a protein other than a p63protein, as defined herein. In preferred embodiments, theoligonucleotide probe detects only a p63 gene, e.g., it does notsubstantially hybridize to transcripts encoding either p53 or p73, orcomplements thereof.

[0131] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. In preferred embodiments, transcription of one of the p63 genesis under the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring forms of p63 polypeptide.

[0132] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., via an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a p63 polypeptide or,in the case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the p63 polypeptide isdisrupted.

[0133] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one of the p63 polypeptides, or an antisensetranscript thereto) which has been introduced into a cell. A transgenecould be partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can also be present in a cell in the form of anepisome. A transgene can include one or more transcriptional regulatorysequences and any other nucleic acid, such as introns, that may benecessary for optimal expression of a selected nucleic acid.

[0134] A “transgenic animal” refers to any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the p63 polypeptide, e.g. either agonistic orantagonistic forms. However, transgenic animals in which the recombinantp63 gene is silent are also contemplated, as for example, the FLP or CRErecombinase dependent constructs described below. Moreover, “transgenicanimal” also includes those recombinant animals in which gene disruptionof one or more p63 genes is caused by human intervention, including bothrecombination and antisense techniques.

[0135] The term “treating” as used herein is intended to encompasscuring as well as ameliorating at least one symptom of the condition ordisease.

[0136] The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

[0137] The term “wild-type allele” refers to an allele of a gene which,when present in two copies in a subject results in a wild-typephenotype. There can be several different wild-type alleles of aspecific gene, since certain nucleotide changes in a gene may not affectthe phenotype of a subject having two copies of the gene with thenucleotide changes.

[0138] 4.3. Nucleic Acids of the Present Invention

[0139] As described below, one aspect of the invention pertains toisolated nucleic acids comprising a nucleotide sequence encoding p63polypeptides, variants and/or equivalents of such nucleic acids.

[0140] Preferred nucleic acids including coding sequences fromvertebrate p63 gene, especially a mammalian p63 gene. Regardless of thespecies, particularly preferred p63 nucleic acids encode polypeptidesthat are at least 70%, 75%, 80%, 90%, 95%, 97%, or 98% similar to anamino acid sequence of a vertebrate p63 protein. In one embodiment, thenucleic acid is a cDNA encoding a polypeptide having at least onebio-activity of the subject p63 polypeptide. Preferably, the nucleicacid includes all or a portion of the nucleotide sequence correspondingto the nucleic acid of SEQ ID Nos 1-12.

[0141] Still other preferred nucleic acids of the present inventionencode a p63 polypeptide which is comprised of at least 2, 5, 10, 25,50, 100, 150 or 200 contiguous amino acid residues. For example,preferred nucleic acid molecules for use as probes/primer or antisensemolecules (i.e. noncoding nucleic acid molecules) can comprise at leastabout 6, 12, 20, 30, 50, 60, 70, 80, 90 or 100 base pairs in length,whereas coding nucleic acid molecules can comprise about 50, 60, 70, 80,90, or 100 base pairs.

[0142] In yet another embodiment, the fragment includes the DNA bindingdomain of p63 and comprises at least 5 contiguous amino acid residues ofSEQ ID Nos. 13-24; the fragment includes the DNA binding domain of p63and comprises at least 20 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the DNA binding domain of p63 and comprisesat least 50 contiguous amino acid residues of SEQ ID Nos. 13-24.

[0143] Another aspect of the invention provides a nucleic acid whichhybridizes under low, medium, or high stringency conditions to a nucleicacid sequences represented by SEQ ID NOs: 1, 2, 3, or 4. Appropriatestringency conditions which promote DNA hybridization, for example, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by a washof 2.0×SSC at 50° C., are known to those skilled in the art or can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-12.3.6. For example, the salt concentration in the washstep can be selected from a low stringency of about 2.0×SSC at 50° C. toa high stringency of about 0.2×SSC at 50° C. In addition, thetemperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C., to high stringencyconditions at about 65° C. Both temperature and salt may be varied, ortemperature of salt concentration may be held constant while the othervariable is changed. In a preferred embodiment, a p63 nucleic acid ofthe present invention will bind to one of SEQ ID NOs 1-12 undermoderately stringent conditions, for example at about 2.0×SSC and about40° C. In a particularly preferred embodiment, a p63 nucleic acid of thepresent invention will bind to one of SEQ ID NOs: 1-12 under highstringency conditions.

[0144] Preferred nucleic acids have a sequence at least 70%, and morepreferably 80% identical and more preferably 90% and even morepreferably at least 95% identical to an amino acid sequence of a p63gene, e.g., such as a sequence shown in one of SEQ ID NOS: 13-24.Nucleic acids at least 90%, more preferably 95%, and most preferably atleast about 98-99% identical with a nucleic sequence represented in oneof SEQ ID NOS: 1-12 are of course also within the scope of theinvention. In preferred embodiments, the nucleic acid is mammalian andin particularly preferred embodiments, includes all or a portion of thenucleotide sequence corresponding to the coding region of one of SEQ IDNOs: 1-12.

[0145] Nucleic acids having a sequence that differs from the nucleotidesequences shown in one of SEQ ID NOs: 1-12 due to degeneracy in thegenetic code are also within the scope of the invention. Such nucleicacids encode functionally equivalent peptides (i.e., a peptide having abiological activity of a p63 polypeptide) but differ in sequence fromthe sequence shown in the sequence listing due to degeneracy in thegenetic code. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC each encode histidine) may result in“silent” mutations which do not affect the amino acid sequence of a p63polypeptide. However, it is expected that DNA sequence polymorphismsthat do lead to changes in the amino acid sequences of the subject p63polypeptides will exist among mammals. One skilled in the art willappreciate that these variations in one or more nucleotides (e.g., up toabout 3-5% of the nucleotides) of the nucleic acids encodingpolypeptides having an activity of a p63 polypeptide may exist amongindividuals of a given species due to natural allelic variation.

[0146] Also within the scope of the invention are nucleic acids encodingsplicing variants of p63 proteins or natural homologs of p63 proteinswhich consist essentially of one of the two units of p63. Such homologscan be cloned by hybridization or PCR, as further described herein.

[0147] The polynucleotide sequence may also encode for a leadersequence, e.g., the natural leader sequence or a heterologous leadersequence. For example, the desired DNA sequence may be fused in the samereading frame to a DNA sequence which aids in expression and secretionof the polypeptide from the host cell, for example, a leader sequencewhich functions as a secretory sequence for controlling transport of thepolypeptide from the cell. The protein having a leader sequence is apreprotein and may have the leader sequence cleaved by the host cell toform the mature form of the protein.

[0148] The polynucleotide of the present invention may also be fused inframe to a marker sequence, also referred to herein as “Tag sequence”encoding a “Tag peptide”, which allows for marking and/or purificationof the polypeptide of the present invention. In a preferred embodiment,the marker sequence is a hexahistidine tag, e.g., supplied by a PQE-9vector. Numerous other Tag peptides are available commercially. Otherfrequently used Tags include myc-epitopes (e.g., see Ellison et al.(1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequencefrom c-myc, the pFLAG system (International Biotechnologies, Inc.), thepEZZ-protein A system (Pharmacia, NJ), and a 16 amino acid portion ofthe Haemophilus influenza hemagglutinin protein. Furthermore, anypolypeptide can be used as a Tag so long as a reagent, e.g., an antibodyinteracting specifically with the Tag polypeptide is available or can beprepared or identified.

[0149] As indicated by the examples set out below, p63 protein-encodingnucleic acids can be obtained from mRNA present in any of a number ofeukaryotic cells, e.g., and is preferably obtained from metazoan cells,more preferably from vertebrate cells and even more preferably frommammalian cells. It should also be possible to obtain nucleic acidsencoding p63 polypeptides of the present invention from genomic DNA fromboth adults and embryos. For example, a gene encoding a p63 protein canbe cloned from either a cDNA or a genomic library in accordance withprotocols described herein, as well as those generally known to personsskilled in the art. cDNA encoding a p63 protein can be obtained byisolating total mRNA from a cell, e.g., a vertebrate cell, a mammaliancell, or a human cell, including embryonic cells. Double stranded cDNAscan then be prepared from the total mRNA, and subsequently inserted intoa suitable plasmid or bacteriophage vector using any one of a number ofknown techniques. The gene encoding a p63 protein can also be clonedusing established polymerase chain reaction techniques in accordancewith the nucleotide sequence information provided by the invention. Apreferred nucleic acid is a cDNA represented by a sequence selected fromthe group consisting of SEQ ID NOs: 1-12.

[0150] Preferred nucleic acids encode a vertebrate p63 polypeptidecomprising an amino acid sequence at least 80% identical, morepreferably 90% identical and most preferably 95% identical with an aminoacid sequence contained in any of SEQ ID Nos: 13-24. Nucleic acids whichencode polypeptides at least about 90%, more preferably at least about95%, and most preferably at least about 98-99% homology with an aminoacid sequence represented in SEQ ID No: 13-24 are also within the scopeof the invention. In one embodiment, the nucleic acid is a cDNA encodinga peptide having at least one activity of the subject vertebrate p63polypeptide. Preferably, the nucleic acid includes all or a portion ofthe nucleotide sequence corresponding to the coding region of SEQ IDNos: 1-12.

[0151] Preferred nucleic acids encode a bioactive fragment of avertebrate p63 polypeptide comprising an amino acid sequence at least80% identical or identical, more preferably 90% identical or identicaland most preferably 95% identical or identical with an amino acidsequence selected from the group consisting of SEQ ID No: 13-24. Forinstance, these bioactive fragments may include the DNA binding domains,transactivation domains, oligomerization domain, etc. Nucleic acidswhich encode polypeptides which are at least about 90%, more preferablyat least about 95%, and most preferably these at least about 98-99%homologous or identical, with an amino acid sequence represented in SEQID No: 13-24 are also within the scope of the invention.

[0152] Preferred bioactive fragments of p63 polypeptides includepolypeptides having one or more of the following biological activities:activity as a tumor suppressor, functions in cell cycle control ofvarious developmental processes, apoptosis, gene expression, modulationof proliferation and differentiation, and tumorigenesis. Assays fordetermining whether given homolog of a p63 exhibits these or otherbiological activities are known in the art and are further describedherein.

[0153] In yet another embodiment, the fragment includes the DNA bindingdomain of p63 and comprises at least 5 contiguous amino acid residues ofSEQ ID Nos. 13-24; the fragment includes the DNA binding domain of p63and comprises at least 20 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the DNA binding domain of p63 and comprisesat least 50 contiguous amino acid residues of SEQ ID Nos. 13-24.

[0154] In yet another embodiment, the fragment includes the core domainof p63 and comprises at least 5 contiguous amino acid residues of SEQ IDNos. 13-24; the fragment includes the core domain of p63 and comprisesat least 20 contiguous amino acid residues of SEQ ID Nos. 13-24; thefragment includes the core domain of p63 and comprises at least 50contiguous amino acid residues of SEQ ID Nos. 13-24.

[0155] 4.3.1 Probes and Primers

[0156] The nucleotide sequences determined from the cloning of p63 genesfrom mammalian organisms will further allow for the generation of probesand primers designed. for identifying and/or cloning p63 homologs inother cell types, e.g., from other tissues, as well as p63 homologs fromother mammalian organisms. For instance, the present invention alsoprovides a probe/primer comprising a substantially purifiedoligonucleotide, which oligonucleotide comprising a nucleotide sequencethat hybridizes under stringent conditions to at least approximately 12,preferably 25, more preferably 40, 50 or 75 consecutive nucleotides ofsense or anti-sense sequence selected from the group consisting of SEQID No: 1-12 or naturally occurring mutants thereof. For instance,primers based on the nucleic acid represented in SEQ ID NOs: 1-12 can beused in PCR reactions to clone p63 homologs.

[0157] In yet another embodiment, the invention provides probes/primerscomprising a substantially purified oligonucleotide comprising anucleotide sequence that hybridizes under moderately stringentconditions to at least approximately 12, 16, 25, 40, 50 or 75consecutive nucleotides sense or antisense sequence selected from thegroup consisting of SEQ ID NOS. 1-12, or naturally occurring mutantsthereof.

[0158] In particular, these probes are useful because they provide amethod for detecting mutations in tumor suppressor genes such as p63,p73, p53 or Rb etc. Nucleic acid probes which are complementary to thewild-type p63 and can form mismatches with mutant p63 genes areprovided, which allow for detection by enzymatic or chemical cleavage orby shifts in electrophonetic mobility.

[0159] Likewise, probes based on the subject p63 sequences can be usedto detect transcripts or genomic sequences encoding the same orhomologous proteins, for use, e.g, in prognostic or diagnostic assays.In preferred embodiments, the probe further comprises a label groupattached thereto and able to be detected, e.g., the label group isselected from amongst radioisotopes, fluorescent compounds, enzymes, andenzyme co-factors.

[0160] 4.3.2 Antisense. Ribozyme and Triplex Techniques

[0161] Another aspect of the invention relates to the use of theisolated nucleic acid in “antisense” therapy. As used herein,“antisense” therapy refers to administration or in situ generation ofoligonucleotide molecules or their derivatives which specificallyhybridize (e.g., bind) under cellular conditions, with the cellular mRNAand/or genomic DNA encoding one or more of the subject p63 proteins soas to inhibit expression of that protein, e.g., by inhibitingtranscription and/or translation. The binding may be by conventionalbase pair complementarity, or, for example, in the case of binding toDNA duplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

[0162] An antisense construct of the present invention can be delivered,for example, as an expression plasmid which, when transcribed in thecell, produces RNA which is complementary to at least a unique portionof the cellular mRNA which encodes a p63 protein. Alternatively, theantisense construct is an oligonucleotide probe which is generated exvivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of ap63 gene. Such oligonucleotide probes are preferably modifiedoligonucleotides which are resistant to endogenous nucleases, e.g.,exonucleases and/or endonucleases, and are therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example, by Van der Krol etal. (1988) BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between the −10 and+10 regions of the p63 nucleotide sequence of interest, are preferred.

[0163] Antisense approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to p63 mRNA. The antisenseoligonucleotides will bind to the p63 mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. In the case of double-stranded antisense nucleic acids, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

[0164] Oligonucleotides that are complementary to the 5′ end of themRNA, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have recently been shown to be effective atinhibiting translation of mRNAs as well. (Wagner, R. 1994. Nature372:333). Therefore, oligonucleotides complementary to either the 5′ or3′ untranslated, non-coding regions of a p63 gene could be used in anantisense approach to inhibit translation of endogenous p63 mRNA.Oligonucleotides complementary to the 5′ untranslated region of the mRNAshould include the complement of the AUG start codon. Antisenseoligonucleotides complementary to mRNA coding regions are less efficientinhibitors of translation but could also be used in accordance with theinvention. Whether designed to hybridize to the 5′, 3′ or coding regionof p63 mRNA, antisense nucleic acids should be at least six nucleotidesin length, and are preferably less that about 100 and more preferablyless than about 50, 25, 17 or 10 nucleotides in length.

[0165] Regardless of the choice of target sequence, it is preferred thatin vitro studies are first performed to quantitate the ability of theantisense oligonucleotide to quantitate the ability of the antisenseoligonucleotide to inhibit gene expression. It is preferred that thesestudies utilize controls that distinguish between antisense geneinhibition and nonspecific biological effects of oligonucleotides. It isalso preferred that these studies compare levels of the target RNA orprotein with that of an internal control RNA or protein. Additionally,it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

[0166] The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. the oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

[0167] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxytiethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,

[0168] 5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0169] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0170] The antisense oligonucleotide can also contain a neutralpeptide-like backbone. Such molecules are termed peptide nucleic acid(PNA)-oligomers and are described, e.g., in Perry-O'Keefe et al. (1996)Proc. Natl. Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993) Nature365:566. One advantage of PNA oligomers is their capability to bind tocomplementary DNA essentially independently from the ionic strength ofthe medium due to the neutral backbone of the DNA. In yet anotherembodiment, the antisense oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

[0171] In yet a further embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-12148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0172] Oligonucleotides of the invention may be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate olgonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0173] While antisense nucleotides complementary to the p63 codingregion sequence can be used, those complementary to the transcribeduntranslated region and to the region comprising the initiatingmethionine are most preferred.

[0174] The antisense molecules can be delivered to cells which expressp63 in vivo. A number of methods have been developed for deliveringantisense DNA or RNA to cells; e.g., antisense molecules can be injecteddirectly into the tissue site, or modified antisense molecules, designedto target the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systematically.

[0175] However, it is often difficult to achieve intracellularconcentrations of the antisense sufficient to suppress translation onendogenous mRNAs. Therefore a preferred approach utilizes a recombinantDNA construct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. The use of such aconstruct to transfect target cells in the patient will result in thetranscription of sufficient amounts of single stranded RNAs that willform complementary base pairs with the endogenous p63 transcripts andthereby prevent translation of the p63 mRNA. For example, a vector canbe introduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bemoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al, 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site; e.g.,the choroid plexus or hypothalamus. Alternatively, viral vectors can beused which selectively infect the desired tissue; (e.g., for brain,herpesvirus vectors may be used), in which case administration may beaccomplished by another route (e.g., systematically).

[0176] Ribozyme molecules designed to catalytically cleave p63 mRNAtranscripts can also be used to prevent translation of p63 mRNA andexpression of p63 (See, e.g., PCT International Publication WO90/11364,published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225 andU.S. Pat. No. 5,093,246). While ribozymes that cleave mRNA at sitespecific recognition sequences can be used to destroy p63 mRNAs, the useof hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAsat locations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.There are a number of potential hammerhead ribozyme cleavage siteswithin the nucleotide sequence of human p63 cDNA (FIG. 1). Preferablythe ribozyme is engineered so that the cleavage recognition site islocated near the 5′ end of the p63 mRNA; i.e., to increase efficiencyand minimize the intracellular accumulation of non-functional mRNAtranscripts.

[0177] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in a p63 gene.

[0178] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g., for improved stability, targeting,etc.) and should be delivered to cells which express the p63 gene invivo. A preferred method of delivery involves using a DNA construct“encoding” the robozyme under the control of a strong constitutive polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous p63 messagesand inhibit translation. Because ribozymes unlike antisense molecules,are catalytic, a lower intracellular concentration is required forefficiency.

[0179] Endogenous p63 gene expression can also be reduced byinactivating or “knocking out” the p63 gene or its promoter usingtargeted homologous recombination. (E.g., see Smithies et al., 1985,Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompsonet al., 1989 Cell 5:313-321; each of which is incorporated by referenceherein in its entirety). For example, a mutant, non-functional p63 (or acompletely unrelated DNA sequence) flanked by DNA homologous to theendogenous p63 gene (either the coding regions or regulatory regions ofthe p63 gene) can be used, with or without a selectable marker and/or anegative selectable marker, to transfect cells that express p63 in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the p63 gene. Such approaches areparticularly suited in the agricultural field where modifications to ES(embryonic stem) cells can be used to generate animal offspring with aninactive p63 (e.g., see Thomas & Capecchi 1987 and Thompson 1989,supra). However this approach can be adapted for use in humans providedthe recombinant DNA constructs are directly administered or targeted tothe required site in vivo using appropriate viral vectors, e.g., herpesvirus vectors for delivery to brain tissue; e.g., the hypothalamusand/or choroid plexus.

[0180] Alternatively, endogenous p63 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the p63 gene (i.e., the p63 promoter and/or enhancers) to formtriple helical structures that prevent transcription of the p63 gene intarget cells in the body. (See generally, Helene, C. 1991, AnticancerDrug Des., 6(6):569-84; Helene, C., et al., 1992, Ann, N.Y. Accad. Sci.,660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).

[0181] Nucleic acid molecules to be used in triple helix formation forthe inhibition of transcription are preferably single stranded andcomposed of deoxyribonucleotides. The base composition of theseoligonucleotides should promote triple helix formation via Hoogsteenbase pairing rules, which generally require sizable stretches of eitherpurines or pyrimidines to be present on one strand of a duplex.Nucleotide sequences may be pyrimidine-based, which will result in TATand CGC triplets across the three associated strands of the resultingtriple helix. The pyrimidine-rich molecules provide base complementarityto a purine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

[0182] Alternatively, the potential sequences that can be targeted fortriple helix formation may be increased by creating a so called“switchback” nucleic acid molecule. Switchback molecules are synthesizedin an alternating 5′-3′,3′-5′ manner, such that they base pair withfirst one strand of a duplex and then the other, eliminating thenecessity for a sizable stretch of either purines or pyrimidines to bepresent on one strand of a duplex.

[0183] Antisense RNA and DNA, ribozyme, and triple helix molecules ofthe invention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

[0184] Moreover, various well-known modifications to nucleic acidmolecules may be introduced as a means of increasing intracellularstability and half-life. Possible modifications include but are notlimited to the addition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

[0185] 4.3.3. Vectors Encoding p63 Proteins and p63 Expressing Cells

[0186] The invention further provides plasmids and vectors encoding anp63 protein, which can be used to express an p63 protein in a host cell.The host cell may be any prokaryotic or eukaryotic cell. Thus, anucleotide sequence derived from the cloning of mammalian p63 proteins,encoding all or a selected portion of the full-length protein, can beused to produce a recombinant form of an p63 polypeptide via microbialor eukaryotic cellular processes. Ligating the polynucleotide sequenceinto a gene construct, such as an expression vector, and transforming ortransfecting into hosts, either eukaryotic (yeast, avian, insect ormammalian) or prokaryotic (bacterial cells), are standard procedureswell known in the art.

[0187] Vectors that allow expression of a nucleic acid in a cell arereferred to as expression vectors. Typically, expression vectors usedfor expressing an p63 protein contain a nucleic acid encoding an p63polypeptide, operably linked to at least one transcriptional regulatorysequence. Regulatory sequences are art-recognized and are selected todirect expression of the subject p63 proteins. Transcriptionalregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). In one embodiment, the expression vector includes a recombinantgene encoding a peptide having an agonistic activity of a subject p63polypeptide, or alternatively, encoding a peptide which is anantagonistic form of an p63 protein.

[0188] Suitable vectors for the expression of a p63 polypeptide includeplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

[0189] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, andYRP17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, a p63 polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the p63 genes represented in SEQ ID NOs: 1or 3.

[0190] The preferred mammalian expression vectors contain bothprokaryotic sequences, to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook,Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989)Chapters 16 and 17.

[0191] In some instances, it may be desirable to express the recombinantp63 polypeptide by the use of a baculovirus expression system. Examplesof such baculovirus expression systems include pVL-derived vectors (suchas pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III)

[0192] When it is desirable to express only a portion of a p63 protein,such as a form lacking a portion of the N-terminus, i.e. a truncationmutant which lacks the signal peptide, it may be necessary to add astart codon (ATG) to the oligonucleotide fragment containing the desiredsequence to be expressed. It is well known in the art that a methionineat the N-terminal position can be enzymatically cleaved by the use ofthe enzyme methionine aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing p63 derived polypeptides in a hostwhich produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitroby use of purified MAP (e.g., procedure of Miller et al., supra).

[0193] Moreover, the gene constructs of the present invention can alsobe used as part of a gene therapy protocol to deliver nucleic acidsencoding either an agonistic or antagonistic form of one of the subjectp63 proteins. Thus, another aspect of the invention features expressionvectors for in vivo or in vitro transfection and expression of a p63polypeptide in particular cell types so as to reconstitute the functionof, or alternatively, abrogate the function of p63 in a tissue. Thiscould be desirable, for example, when the naturally-occurring form ofthe protein is misexpressed or the natural protein is mutated and lessactive.

[0194] In addition to viral transfer methods, non-viral methods can alsobe employed to cause expression of a subject p63 polypeptide in thetissue of an animal. Most nonviral methods of gene transfer rely onnormal mechanisms used by mammalian cells for the uptake andintracellular transport of macromolecules. In preferred embodiments,non-viral targeting means of the present invention rely on endocyticpathways for the uptake of the subject p63 polypeptide gene by thetargeted cell. Exemplary targeting means of this type include liposomalderived systems, poly-lysine conjugates, and artificial viral envelopes.

[0195] In other embodiments transgenic animals, described in more detailbelow could be used to produce recombinant proteins.

[0196] 4.4. Polypeptides of the Present Invention

[0197] The present invention makes available isolated p63 polypeptideswhich are isolated from, or otherwise substantially free of othercellular proteins, especially other signal transduction factors and/ortranscription factors which may normally be associated with the p63polypeptide. The term “substantially free of other cellular proteins”(also referred to herein as “contaminating proteins”) or “substantiallypure or purified preparations” are defined as encompassing preparationsof p63 polypeptides having less than about 20% (by dry weight)contaminating protein, and preferably having less than about 5%contaminating protein. Functional forms of the subject polypeptides canbe prepared, for the first time, as purified preparations by using acloned gene as described herein. Full length proteins or fragmentscorresponding to one or more particular motifs and/or domains or toarbitrary sizes, for example, at least 5, 10, 25, 50, 75 and 100, aminoacids in length are within the scope of the present invention.

[0198] In yet another embodiment, the fragment includes the core domainof p63 and comprises at least 5 contiguous amino acid residues of SEQ IDNos. 13-24; the fragment includes the core domain of p63 and comprisesat least 20 contiguous amino acid residues of SEQ ID Nos. 13-24; thefragment includes the core domain of p63 and comprises at least 50contiguous amino acid residues of SEQ ID Nos. 13-24.

[0199] In yet another embodiment, the fragment includes the DNA bindingdomain of p63 and comprises at least 5 contiguous amino acid residues ofSEQ ID Nos. 13-24; the fragment includes the DNA binding of p63 andcomprises at least 20 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the DNA binding of p63 and comprises atleast 50 contiguous amino acid residues of SEQ ID Nos. 13-24.

[0200] For example, isolated p63 polypeptides can be encoded by all or aportion of a nucleic acid sequence shown in any of SEQ ID NOS. 1-12.Isolated peptidyl portions of p63 proteins can be obtained by screeningpeptides recombinantly produced from the corresponding fragment of thenucleic acid encoding such peptides. In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Forexample, a p63 polypeptide of the present invention may be arbitrarilydivided into fragments of desired length with no overlap of thefragments, or preferably divided into overlapping fragments of a desiredlength. The fragments can be produced (recombinantly or by chemicalsynthesis) and tested to identify those peptidyl fragments which canfunction as either agonists or antagonists of a wild-type (e.g.,“authentic”) p63 protein.

[0201] Another aspect of the present invention concerns recombinantforms of the p63 proteins. Recombinant polypeptides preferred by thepresent invention, in addition to native p63 proteins (e.g., as setforth in SEQ ID NO: 6), are encoded by a nucleic acid, which is at least60%, more preferably at least 80%, and more preferably 85%, and morepreferably 90%, and more preferably 95% identical to an amino acidsequence represented by SEQ ID Nos: 13-24 or encoded by SEQ ID NOs.13-24. Polypeptides which are encoded by a nucleic acid that is at leastabout 98-99% identical with the sequence of SEQ ID NOS: 1 or 3 or whichare 98-99% identical with the amino acid sequence set forth in SEQ IDNO: 2 are also within the scope of the invention.

[0202] In a preferred embodiment, a p63 protein of the present inventionis a mammalian p63 protein and even more preferably a human p63 protein.In a particularly preferred embodiment the p63 protein has an amino acidsequence as set forth in SEQ ID Nos: 13-24. In particularly preferredembodiment, the p63 protein retains p63 bioactivity. It will beunderstood that certain post-translational modifications, e.g.,phosphorylation and the like, can increase the apparent molecular weightof the p63 protein relative to the unmodified polypeptide chain.

[0203] The present invention further pertains to recombinant forms ofone of the subject p63 polypeptides. Such recombinant p63 polypeptidespreferably are capable of functioning in one of either role of anagonist or antagonist of at least one biological activity of a wild-type(“authentic”) p63 protein of the appended sequence listing. The term“evolutionarily related to”, with respect to amino acid sequences of p63proteins, refers to both polypeptides having amino acid sequences whichhave arisen naturally, and also to mutational variants of human p63polypeptides which are derived, for example, by combinatorialmutagenesis.

[0204] In general, polypeptides referred to herein as having an activity(e.g., are “bioactive”) of a p63 protein are defined as polypeptideswhich include an amino acid sequence encoded by all or a portion of thenucleic acid sequences shown in one of SEQ ID NOS: 1-12 and which mimicor antagonize all or a portion of the biological/biochemical activitiesof a naturally occurring p63 protein. Preferred bioactive fragments ofp63 polypeptides include polypeptides having one or more of thefollowing biological activities: activity as a tumor suppressor,functions in cell cycle control various developmental processes,apoptosis, gene expression and tumorigenesis. Other biologicalactivities of the subject p63 proteins are described herein or will bereasonably apparent to those skilled in the art. According to thepresent invention, a polypeptide has biological activity if it is aspecific agonist or antagonist of a naturally-occurring form of a p63protein.

[0205] Assays for determining whether a compound, e.g, a protein, suchas an p63 protein or variant thereof, has one or more of the abovebiological activities are well known in the art.

[0206] In another embodiment, the coding sequences for the polypeptidecan be incorporated as a part of a fusion gene including a nucleotidesequence encoding a different polypeptide. This type of expressionsystem can be useful under conditions where it is desirable to producean immunogenic fragment of a p63 protein. For example, the VP6 capsidprotein of rotavirus can be used as an immunologic carrier protein forportions of the p63 polypeptide, either in the monomeric form or in theform of a viral particle. The nucleic acid sequences corresponding tothe portion of a subject p63 protein to which antibodies are to beraised can be incorporated into a fusion gene construct which includescoding sequences for a late vaccinia virus structural protein to producea set of recombinant viruses expressing fusion proteins comprising p63epitopes as part of the virion. It has been demonstrated with the use ofimmunogenic fusion proteins utilizing the Hepatitis B surface antigenfusion proteins that recombinant Hepatitis B virions can be utilized inthis role as well. Similarly, chimeric constructs coding for fusionproteins containing a portion of a p63protein and the poliovirus capsidprotein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see, for example, EP Publication No: 0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

[0207] The Multiple antigen peptide system for peptide-basedimmunization can also be utilized to generate an immunogen, wherein adesired portion of a p63 polypeptide is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see, for example, Posnett et al. (1988) JBC 263:1719 andNardelli et al. (1992) J. Immunol. 148:914). Antigenic determinants ofp63 proteins can also be expressed and presented by bacterial cells.

[0208] In addition to utilizing fusion proteins to enhanceimmunogenicity, it is widely appreciated that fusion proteins can alsofacilitate the expression of proteins, and accordingly, can be used inthe expression of the p63 polypeptides of the present invention. Forexample, p63 polypeptides can be generated as glutathione-S-transferase(GST-fusion) proteins. Such GST-fusion proteins can enable easypurification of the p63 polypeptide, as for example by the use ofglutathione-derivatized matrices (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).

[0209] In another embodiment, a fusion gene coding for a purificationleader sequence, such as a poly-(His)/enterokinase cleavage sitesequence at the N-terminus of the desired portion of the recombinantprotein, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified protein (e.g., see Hochuli et al.(1987) J. Chromatography 411:177; and Janknecht et al. PNAS 88:8972).Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

[0210] The present invention further pertains to methods of producingthe subject p63 polypeptides. For example, a host cell transfected witha nucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. Suitable mediafor cell culture are well known in the art. The recombinant p63polypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In a preferred embodiment, therecombinant p63 polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as GST fusion protein.

[0211] Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject p63 polypeptides which function in a limited capacity as one ofeither a p63 agonist (mimetic) or a p63 antagonist, in order to promoteor inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homolog of limited function,and with fewer side effects relative to treatment with agonists orantagonists which are directed to all of the biological activities ofnaturally occurring forms of p63 proteins.

[0212] Homologs of each of the subject p63 proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the p63 polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an p63 receptor.

[0213] The recombinant p63 polypeptides of the present invention alsoinclude homologs of the wildtype p63 proteins, such as versions of thoseprotein which are resistant to proteolytic cleavage, as for example, dueto mutations which alter ubiquitination or other enzymatic targetingassociated with the protein.

[0214] p63 polypeptides may also be chemically modified to create p63derivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of p63 proteins can beprepared by linking the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

[0215] Modification of the structure of the subject p63 polypeptides canbe for such purposes as enhancing therapeutic or prophylactic efficacy,stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation), or post-translational modifications (e.g., to alterphosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the p63 polypeptides described inmore detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition. Thesubstitutional variant may be a substituted conserved amino acid or asubstituted non-conserved amino acid.

[0216] For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. isosteric and/orisoelectric mutations) will not have a major effect on the biologicalactivity of the resulting molecule. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2^(nd) ed., Ed. by L.Stryer, W H Freeman and Co.: 1981). Whether a change in the amino acidsequence of a peptide results in a functional p63 homolog (e.g.,functional in the sense that the resulting polypeptide mimics orantagonizes the wild-type form) can be readily determined by assessingthe ability of the variant peptide to produce a response in cells in afashion similar to the wild-type protein, or competitively inhibit sucha response. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner.

[0217] This invention further contemplates a method for generating setsof combinatorial mutants of the subject p63 proteins as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (e.g., homologs). The purpose of screening suchcombinatorial libraries is to generate, for example, novel p63 homologswhich can act as either agonists or antagonist, or alternatively,possess novel activities all together. Thus, combinatorially-derivedhomologs can be generated to have an increased potency relative to anaturally occurring form of the protein.

[0218] In one embodiment, the variegated library of p63 variants isgenerated by combinatorial mutagenesis at the nucleic acid level, and isencoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential p63 sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofp63 sequences therein. There are many ways by which such libraries ofpotential p63 homologs can be generated from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be carried out in an automatic DNA synthesizer, and thesynthetic genes then ligated into an appropriate expression vector. Thepurpose of a degenerate set of genes is to provide, in one mixture, allof the sequences encoding the desired set of potential p63 sequences.The synthesis of degenerate oligonucleotides is well known in the art(see for example, Narang, S A (1983) Tetrahedron 39:3; Itakura et al.(1981) Recombinant DNA, Proc 3^(rd) Cleveland Sympos. Macromolecules,ed. A G Walton, Amsterdam: Elsevier pp 273-289; Itakura et al. (1984)Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ikeet al. (1983) Nucleic Acid Res. 11:477. Such techniques have beenemployed in the directed evolution of other proteins (see, for example,Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al.(1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

[0219] Likewise, a library of coding sequence fragments can be providedfor a p63 clone in order to generate a variegated population of p63fragments for screening and subsequent selection of bioactive fragments.A variety of techniques are known in the art for generating suchlibraries, including chemical synthesis. In one embodiment, a library ofcoding sequence fragments can be generated by (i) treating a doublestranded PCR fragment of a p63 coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule; (ii)denaturing the double stranded DNA; (iii) renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products; (iv) removing single stranded portions fromreformed duplexes by treatment with S1 nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes.

[0220] A wide range of techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of p63 homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate p63 sequences created bycombinatorial mutagenesis techniques. Combinatorial mutagenesis has apotential to generate very large libraries of mutant proteins, e.g., inthe order of 1026 molecules. Combinatorial libraries of this size may betechnically challenging to screen even with high throughput screeningassays. To overcome this problem, a new technique has been developedrecently, recrusive ensemble mutagenesis (REM), which allows one toavoid the very high proportion of non-functional proteins in a randomlibrary and simply enhances the frequency of functional proteins, thusdecreasing the complexity required to achieve a useful sampling ofsequence space. REM is an algorithm which enhances the frequency offunctional mutants in a library when an appropriate selection orscreening method is employed (Arkin and Yourvan, 1992, PNAS USA89:7811-7815; Yourvan et al., 1992, Parallel Problem Solving fromNature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co.,Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering6(3):327-331).

[0221] The invention also provides for reduction of the p63 proteins togenerate mimetics, e.g., peptide or non-pepide agents, such as smallmolecules, which are able to disrupt binding of a p63 polypeptide of thepresent invention with a nucleotide, such as proteins, e.g. receptors.Thus, such mutagenic techniques as described above are also useful tomap the determinants of the p63 proteins which participate inprotein-protein interactions involved in, for example, binding of thesubject p63 polypeptide to a target peptide. To illustrate, the criticalresidues of a subject p63 polypeptide which are involved in molecularrecognition of its receptor can be determined and used to generate p63derived peptidomimetics or small molecules which competitively inhibitbinding of the authentic p63 protein with that moiety. By employing, forexample, scanning mutagenesis to map the amino acid residues of thesubject p63 proteins which are involved in binding other proteins,peptidomimetic compounds can be generated which mimic those residues ofthe p63 protein which facilitate the interaction. Such mimetics may thenbe used to interfere with the normal function of a p63 protein. Forinstance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gamma lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9^(th) American Peptide Symposium)Pierce Chemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagaiet al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem SocPerkin Trans 1:1231), and β-aminoalcohols (Gordon et al. (1985) BiochemBiophys Res Commun 126:419; and Dann et al. (1986) Biochem Biophys ResCommun 134:71).

[0222] 4.5. Anti-63 Antibodies and Uses Therefor

[0223] Another aspect of the invention pertains to an antibodyspecifically reactive with a mammalian p63 protein, e.g., a wild-type ormutated p63 protein. For example antibodies may be made as described inteh appended rxamples or by using other standard protocols (See, forexample, Antibodies: A Laboratory Manual ed. by Harlow and Lane (ColdSpring Harbor Press: 1988)). A mammal, such as a mouse, a hamster orrabbit can be immunized with an immunogenic form of the peptide (e.g., amammalian p63 polypeptide or an antigenic fragment which is capable ofeliciting an antibody response, or a fusion protein as described above).

[0224] In one aspect, this invention includes monoclonal antibodies top63 that show p63 is highly expressed in the basal cells of variousepithelial tissues, including epidermis, ectocervical epithelium,vaginal epithelium, urothelium, and prostate epthelium, all of whichrepresent common sites of human carcinomas (basal cell carcinoma ofskin, cervical carcinoma with and without human papilloma virusassociation, bladder and urothelial carcinoma, and prostate carcinoma).Therefore, in one embodiment this invention provides a diagnostic toolfor the analysis of p63 expression in general, and in particular, as adiagnostic for analysis of carcinomas.

[0225] Techniques for conferring immunogenicity on a protein or peptideinclude conjugation to carriers or other techniques well known in theart. An immunogenic portion of a p63 protein can be administered in thepresence of adjuvant. The progress of immunization can be monitored bydetection of antibody titers in plasma or serum. Standard ELISA or otherimmunoassays can be used with the immunogen as antigen to assess thelevels of antibodies. In a preferred embodiment, the subject antibodiesare immunospecific for antigenic determinants of a p63 protein of amammal, e.g., antigenic determinants of a protein set forth in SEQ IDNo: 2 or closely related homologs (e.g., at least 90% identical, andmore preferably at least 95% identical).

[0226] Following immunization of an animal with an antigenic preparationof a p63 polypeptide, anti-p63 antisera can be obtained and, if desired,polyclonal anti-p63 antibodies isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a mammalian p63polypeptide of the present invention and monoclonal antibodies isolatedfrom a culture comprising such hybridoma cells. In one embodimentanti-human p63 antibodies specifically react with the protein encoded bythe DNA of ATCC deposit No.______.

[0227] The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectmammalian p63 polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific, single-chain, and chimeric and humanizedmolecules having affinity for a p63 protein conferred by at least oneCDR region of the antibody. In preferred embodiments, the antibodies,the antibody further comprises a label attached thereto and able to bedetected, (e.g., the label can be a radioisotope, fluorescent compound,enzyme or enzyme co-factor).

[0228] Anti-p63 antibodies can be used, e.g., to monitor p63 proteinlevels in an individual for determining, e.g., whether a subject has adisease or condition associated with an aberrant p63 protein level, orallowing determination of the efficacy of a given treatment regimen foran individual afflicted with such a disorder. The level of p63polypeptides may be measured from cells in bodily fluid, such as inblood samples.

[0229] Another application of anti-p63 antibodies of the presentinvention is in the immunological screening of cDNA librariesconstructed in expression vectors such as λgt11, λgt18-23, λZAP, andλORF8. Messenger libraries of this type, having coding sequencesinserted in the correct reading frame and orientation, can producefusion proteins. For instance, λgt11 will produce fusion proteins whoseamino termini consist of β-galactosidase amino acid sequences and whosecarboxy termini consist of a foreign polypeptide. Antigenic epitopes ofa p63 protein, e.g., other orthologs of a particular p63 protein orother paralogs from the same species, can then be detected withantibodies, as, for example, reacting nitrocellulose filters lifted frominfected plates with anti-p63 antibodies. Positive phage detected bythis assay can then be isolated from the infected plate. Thus, thepresence of p63 homologs can be detected and cloned from other animals,as can alternate isoforms (including splicing variants) from humans.

[0230] In another embodiment, a panel of monoclonal antibodies may beused, wherein each of the epitopes involved p63 functions arerepresented by a monoclonal antibody. Loss or perturbation of binding ofa monoclonal antibody in the panel would be indicative of a mutationalattention of the p63 protein and thus of the p63 gene.

[0231] 4.6. Transgenic Animals

[0232] One aspect of the present invention relates to transgenicnon-human animals having germline and/or somatic cells in which thebiological activity of one or more tumor supressor genes, e.g., p63,p53, p73 proteins, combinations thereof, are altered by a chromosomallyincorporated transgene.

[0233] In one preferred embodiment, the transgene disrupts at least aportion of a genomic p63 gene. For instance, the transgene may deleteall or a portion of the genomic p63 gene by replacement recombination,or may functionally interrupt one or more of a regulatory sequence orcoding sequence of the genomic p63 gene by insertion recombination.

[0234] In another preferred embodiment, the transgene encodes a p63protein, and expression of the transgene in cells of the transgenicanimal results in altered regulation of the level of the p63 proteinrelative to normal expression of the wild-type p63 protein.

[0235] In still other preferred embodiments, the transgene encodes amutant p63 protein, such as dominant negative p63 protein whichantagonizes at least a portion of the biological function of a wild-typep63 protein.

[0236] Yet another preferred transgenic animal includes a transgeneencoding an antisense transcript which, when transcribed from thetransgene, hybridizes with a genomic p63 gene or a mRNA transcriptthereof, and inhibits expression of the genomic p63 gene.

[0237] In one embodiment, the present invention provides a desirednon-human animal or an animal (including human) cell which contains apredefined, specific and desired alteration rendering the non-humananimal or animal cell predisposed to cancer. Specifically, the inventionpertains to a genetically altered non-human animal (most preferably, amouse), or a cell (either non-human animal or human) in culture, that isdefective in at least one of two alleles of a tumor-suppressor gene suchas the p63 gene. The inactivation of at least one of these tumorsuppressor alleles results in an animal with a higher susceptibility totumor inductionor other proliferative or differentiative disorders, ordisorders marked by abberrant signal transduction, e/g/, from a cytokineor growth factor. A genetically altered mouse of this type is able toserve as a useful model for hereditary cancers and as a test animal forcarcinogen studies. The invention additionally pertains to the use ofsuch non-human animals or animal cells, and their progeny in researchand medicine.

[0238] Furthermore, it is contemplated that cells of the transgenicanimals of the present invention can include other transgenes, e.g.,which alter the biological activity of a second tumor suppressor gene oran oncogene. For instance, the second transgene can functionally disruptthe biological activity of a second tumor suppressor gene, such as p53,p73, DCC, p21^(cip1), p27^(kip1), Rb, Mad or E2F. Alternatively, thesecond transgene can cause overexpression or loss of regulation of anoncogene, such as ras, myc, a cdc25 phosphatase, Bcl-2, Bcl-6, atransforming growth factor, neu, int-3, polyoma virus middle T antigen,SV40 large T antigen, a papillomaviral E6 protein, a papillomaviral E7protein, CDK4, or cyclin D1.

[0239] A preferred transgenic non-human animal of the present inventionhas germline and/or somatic cells in which one or more alleles of agenomic p63 gene, a p73 gene, a p53 gene, and combinations thereof, aredisrupted by a chromosomally incorporated transgene, wherein thetransgene includes a marker sequence providing a detectable signal foridentifying the presence of the transgene in cells of the transgenicanimal, and replaces at least a portion of the genomic p63 gene or isinserted into the genomic p63 gene or disrupt expression of a wild typep63 protein.

[0240] Another aspect of the present invention relates to cells andtissues isolated from the subject transgenic animals. For instance, thepresent invention provides composition of cells, isolated ex vivo, whichinclude a diploid genome having a chromosomally incorporated transgene,which transgene functionally modifies the biological activity of one ormore p63 proteins. In preferred embodiments, the transgene deletes allor a portion of a genomic p63 gene by replacement recombination, orfunctionally interrupts one or more of a regulatory sequence or codingsequence of the genomic p63 gene by insertion recombination. Forinstance, one class of such cells contemplated by the present inventioninclude transgenes which have (i) at least a portion of the genomic p63gene which directs recombination of the transgene with the genomic p63gene, and (ii) a marker sequence which provides a detectable signal foridentifying the presence of the transgene in a cell.

[0241] The animals of this invention can be used as a source of cells,differentiated or precursor, which can be immortalized in cell culture.In a preferred embodiment, the cells are stem cells or pluripotentprogenitor cells. For instance, such cells can be precursors ofhematopoietic cells, neuronal cells, pancreatic cells, hepatic cells,chondrocytes, osteocytes, myocytes, or combinations thereof.

[0242] Still another aspect of the present invention relates to methodsfor generating non-human animals and stem cells having a functionallydisrupted endogenous p63 gene. In a preferred embodiment, the methodcomprises the steps of:

[0243] (i) constructing a transgene construct including (a) arecombination region having at least a portion of the p63 gene, whichrecombination region directs recombination of the transgene with the p63gene, and (b) a marker sequence which provides a detectable signal foridentifying the presence of the transgene in a cell;

[0244] (ii) transfering the transgene into stem cells of a non-humananimal;

[0245] (iii) selecting stem cells having a correctly targeted homologousrecombination between the transgene and the p63 gene;

[0246] (iv) transfering cells identified in step (iii) into a non-humanblastocyst and implanting the resulting chimeric blastocyst into anon-human female; and

[0247] (v) collecting offspring harboring an endogenous p63 gene allelehaving the correctly targeted recombinantion.

[0248] Yet another aspect of the invention provides a method forevaluating the carcinogenic potential of an agent by (i) contacting atransgenic animal of the present invention with a test agent, and (ii)comparing the number of transformed cells in a sample from the treatedanimal with the number of transformed cells in a sample from anuntreated transgenic animal or transgenic animal treated with a controlagent. The difference in the number of transformed cells in the treatedanimal, relative to the number of transformed cells in the absence oftreatment with a control agent, indicates the carcinogenic potential ofthe test compound.

[0249] Another aspect of the invention provides a method of evaluatingan anti-proliferative activity of a test compound In preferredembodiments, the method includes contacting a transgenic animal of thepresent invention, or a sample of cells from such animal, with a testagent, and determining the number of transformed cells in a specimenfrom the transgenic animal or in the sample of cells. A statisticallysignificant decrease in the number of transformed cells, relative to thenumber of transformed cells in the absence of the test agent, indicatesthe test compound is a potential anti-proliferative agent.

[0250] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. 1. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0251] 4.7 Screening Assays for p63 Therapeutics

[0252] The invention provides for p63 therapeutic compounds for treatingdiseases or conditions caused by, or contributed to by an abnormal p63activity, e.g., a predisposition to form tumors. The compounds that canbe used for this purpose can be any type of compound, including aprotein, a peptide, peptidomimetic, small molecule, and nucleic acid. Anucleic acid can be, e.g., a gene, an antisense nucleic acid, aribozyme, or a triplex molecule. A compound of the invention can be anagonist or an antagonist. Preferred p63 agonists include p63 proteins orderivatives thereof which mimic at least one p63 activity, e.g., theability to activate transcription, act as a tumor suppressor, inhibittumorigenesis by eliminating potentially tumorigeale cells hydrolysis ofa target peptide or nucleic acids encoding such. Other preferredagonists include compounds which are capable of increasing theproduction of p63 protein in cells, e.g., compounds capable ofupregulating the expression of a p63 gene. Preferred p63 antagonistsinclude compounds which decrease or inhibit interaction of a p63 proteinwith a target gene. In a preferred embodiment, a p63 antagonist is amodified form of a target peptide, which is capable of interacting withthe target gene, but which does not have biological activity, e.g., willnot act as a transcription factor.

[0253] It is possible that when p63 functions as a transcription factor,it uses its central domain to bind to its target sequence. This regionof p63 may also b a target for other proteins that interact with it.These proteins, could increase or decrease transactivation. Accordingly,compounds modulating the interaction of such proteins with p63 could beagonists or antagonists.

[0254] Thus, the invention provides methods for identifying p63 agonistand antagonist compounds, comprising selecting compounds which arecapable of modulating the interaction of an p63 protein with anothermolecule referred to herein as “p63 binding partner”. A p63 bindingpartner can be a target gene or a target oncoprotein etc. A p63 bindingpartner can also be a polypeptide which is not a target peptide andwhich may, e.g., interact with a p63 protein at sites other than itsmajor binding domain. In yet other embodiments of the invention, an p63therapeutic is a compound which is capable of binding to a p63 protein,e.g., a wild-type p63 protein or a mutated form of a p63 protein, andthereby modulate the catalytic activity of the p63-protein or degrade orcause the p63 protein to be degraded. For example, such an p63therapeutic can be an antibody or derivative thereof which interactsspecifically with an p63 protein (either wild-type or mutated).

[0255] In a further embodiment, the p63 therapeutic of the invention iscapable of acting on an p63 gene, e.g., to modulate its expression.

[0256] The compounds of the invention can be identified using variousassays depending on the type of compound and activity of the compoundthat is desired. Set forth below are at least some assays that can beused for identifying p63 therapeutics. It is within the skill of the artto design additional assays for identifying p63 therapeutics.

[0257] 4.7.1 Cell-Free Assays

[0258] Cell-free assays can be used to identify compounds which modulatethe interaction between an p63 protein and a p63 binding partner, suchas a target gene or peptide. In a preferred embodiment, cell-free assaysfor identifying such compounds consist essentially in combining togetherin a reaction mixture a p63 protein, a p63 binding partner and a testcompound or a library of test compounds. A test compound can be aderivative of a p63 binding partner, e.g., an biologically inactivetarget peptide, or the test compound can be a small molecule.

[0259] Accordingly, an exemplary screening assay of the presentinvention includes the steps of (a) forming a reaction mixtureincluding: (i) a p63 polypeptide, (ii) a p63 binding partner (e.g., p21such as a target gene, examples include activation of p21, whichexhibits the cell cycle and/or GADD45 a repair protein activated bypathways that respond to irradiation damage), and (iii) a test compound;and (b) detecting interaction of the p63 and the p63 binding protein.The p63 polypeptide and p63 binding partner can be producedrecombinantly, purified from a source, e.g., plasma, or chemicallysynthesized, as described herein. A statistically significant change(potentiation or inhibition) in the interaction of the p63 and p63binding protein in the presence of the test compound, relative to theinteraction in the absence of the test compound, indicates a potentialagonist (mimetic or potentiator) or antagonist (inhibitor) of p63bioactivity for the test compound. The compounds of this assay can becontacted simultaneously. Alternatively, a p63 protein can first becontacted with a test compound for an appropriate amount of time,following which the p63 binding partner is added to the reactionmixture. The efficacy of the compound can be assessed by generating doseresponse curves from data obtained using various concentrations of thetest compound. Moreover, a control assay can also be performed toprovide a baseline for comparison. In the control assay, isolated andpurified p63 polypeptide or binding partner is added to a compositioncontaining the p63 binding partner or p63 polypeptide, and the formationof a complex is quantitated in the absence of the test compound.

[0260] Complex formation between a p63 protein and a p63 binding partnermay be detected by a variety of techniques. Modulation of the formationof complexes can be quantitated using, for example, detectably labeledproteins such as radiolabeled, fluorescently labeled, or enzymaticallylabeled p63 proteins or p63 binding partners, by immunoassay, or bychromatographic detection.

[0261] Typically, it will be desirable to immobilize either p63 or itsbinding partner to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of p63 to a p63 binding partner, can beaccomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase/p63 (GST/p63) fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe p63 binding partner, e.g. an ³⁵S-labeled p63 binding partner, andthe test compound, and the mixture incubated under conditions conduciveto complex formation, e.g. at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g. beads placedin scintilant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level of p63protein or p63 binding partner found in the bead fraction quantitatedfrom the gel using standard electrophoretic techniques such as describedin the appended examples.

[0262] Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either p63 or itscognate binding partner can be immobilized utilizing conjugation ofbiotin and streptavidin. For instance, biotinylated p63 molecules can beprepared from biotin-NHS(N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with p63can be derivatized to the wells of the plate, and p63 trapped in thewells by antibody conjugation. As above, preparations of a p63 bindingprotein and a test compound are incubated in the p63 presenting wells ofthe plate, and the amount of complex trapped in the well can bequantitated. Exemplary methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the p63binding partner, or which are reactive with p63 protein and compete withthe binding partner; as well as enzyme-linked assays which rely ondetecting an enzymatic activity associated with the binding partner,either intrinsic or extrinsic activity. In the instance of the latter,the enzyme can be chemically conjugated or provided as a fusion proteinwith the p63 binding partner. To illustrate, the p63 binding partner canbe chemically cross-linked or genetically fused with horseradishperoxidase, and the amount of polypeptide trapped in the complex can beassessed with a chromogenic substrate of the enzyme, e.g.3,3′-diamino-benzadine terahydrochloride or 4-chloro-1-napthol.Likewise, a fusion protein comprising the polypeptide andglutathione-S-transferase can be provided, and complex formationquantitated by detecting the GST activity usingl-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

[0263] For processes which rely on immunodetection for quantitating oneof the proteins trapped in the complex, antibodies against the protein,such as anti-p63 antibodies, can be used. Alternatively, the protein tobe detected in the complex can be “epitope tagged” in the form of afusion protein which includes, in addition to the p63 sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, NJ).

[0264] Cell-free assays can also be used to identify compounds whichinteract with a p63 protein and modulate an activity of a p63 protein.Accordingly, in one embodiment, a p63 protein is contacted with a testcompound and the actual transcription of a target gene activity of p63is monitored. In one embodiment, the abililty of p63 to bind to and/orto hydrolyze a target peptide, e.g, angiotensin I or a kinin, such asbradykinin is determined. The binding affinity of p63 to a targetpeptide can be determined according to methods known in the art.Determination of the enzymatic activity of p63 can be performed with theaid of the substrate furanacryloyl-L-phenylalanyl-glycyl-glycine (FAPGG)under conditions described in Holmquist et al. (1979) Anal. Biochem.95:540 and in U.S. Pat. No. 5,259,045. The subject screening assays canbe accomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes.

[0265] 4.7.2. Cell Based Assays

[0266] In addition to cell-free assays, such as described above, thereadily available source of p63 proteins provided by the presentinvention also facilitates the generation of cell-based assays foridentifying small molecule agonists/antagonists and the like. Suchassays can be used, e.g., to identify compounds which modulateexpression of a p63 gene, modulate translation of a p63 mRNA, or whichmodulate the stability of a p63 mRNA or protein. Accordingly, in oneembodiment, a cell which is capable of producing p63, [include e.g. ofcells], is incubated with a test compound and the amount of p63 producedin the cell medium is measured and compared to that produced from a cellwhich has not been contacted with the test compound. The specificity ofthe compound vis a vis p63 can be confirmed by various control analysis,e.g., measuring the expression of one or more control gene.

[0267] Compounds which can be tested include small molecules, proteins,and nucleic acids. In particular, this assay can be used to determinethe efficacity of p63 antisense molecules or ribozymes.

[0268] In another embodiment, the effect of a test compound ontranscription of an p63 gene is determined by transfection experimentsusing a reporter gene operatively linked to at least a portion of thepromoter of an p63 gene. A promoter region of a gene can be isolated,e.g., from a genomic library according to methods known in the art. Thereporter gene can be any gene encoding a protein which is readilyquantifiable, e.g, the luciferase or CAT gene, well known in the art.

[0269] In another embodiment, the invention provides a method fordetecting functional p63 protein in cells, preferably mammalian cells.‘functional p63’ means a p63 protein which is able to activate genetranscription. The invention relates to a method of determining thepresence of functional p63 based on the dependence of transactivation ofcertain target genes by p63. Examples include the p21 gene or repairproteins for instance those activated by irradiation damage. The methodcomprises (a) stimulating mammalian cells to increase expression of thetarget mRNA; and (b) coparing the level of the mRNA in stimulated cellsto the level of mRNA in unstimulated cells.

[0270] For instance, primary cultures of mammalian cells can also beused. Such cells can be biopsies taken from mammalian tumors. Mammaliancell cultures can be initiated from biopsies by surgical incisional orescisional methods. In one embodiment, the cells may be stimulated instep (a) by irradiating the cells in order to induce or stimulateexpression of the repair proetins.

[0271] The RNA can be isolated from irradiated mammalian cells bymethods known to those skilled in the art.

[0272] 4.7.3 Ubiquitin-Mediated Proteolysis

[0273] Furthermore, the present invention, by making available purifiedand recombinant forms of the subject p63 proteins, facilitates thedevelopment of assays that can be used to screen for drugs which inhibitthe proteolysis of p63, such as by inhibiting ubiquitination of p63, orubiquitin-mediated proteolysis of p63. For instance, in addition toagents which disrupt binding of p63 to other cellular (or viral)proteins, inhibitors of ubiquitin conjugating enzymes (“E2” enzymes) orubiquitin ligases (“E3” enzymes) may prevent transfer of ubiquitin top63.

[0274] Assays for the measurement of ubiquitination can be generated inmany different forms, and include assays based on cell-free systems,e.g. purified proteins or cell lysates, as well as cell-based assayswhich utilize intact cells. Assays as described herein can be used inconjunction with the subject p63 proteins to generate aubiquitin-conjugating system for detecting agents able to inhibitparticular E2- or E3-mediated ubiquitination of p63 proteins. Suchinhibitors can be used, for example, in the treatment of proliferativeand/or differentiative disorders, to modulate apoptosis, and in thetreatment of viral infections, such by adenoviruses or papillomaviruses.

[0275] In many drug screening programs which test libraries of compoundsand natural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins or with lysates, are oftenpreferred as “primary” screens in that they can be generated to permitrapid development and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with other proteinsor change in enzymatic properties of the molecular target. Accordingly,potential inhibitors of p63 ubiquitination can be detected in acell-free assay generated by consitution of a functionalubiquitin-protein ligase system in a cell lysate, such as generated bycharging a reticulocyte lysate (Hersko et al. (1983)J Biol Chem258:8206-6214) with a p63 polypeptide and, as needed, a specific E1, E2or E3 enzyme (cellular or viral in origin), and ubiquitin. In analternative format, the assay can be derived as a reconstituted proteinmixture.

[0276] In yet other embodiments, the present assay comprises an in vivoubiquitin-conjugating system, such as a cell able to conduct the p63protein through at least a portion of a ubiquitin-mediated proteolyticpathway.

[0277] The level of ubiquitination of the substrate p63 protein broughtabout by the system is measured in the presence and absence of acandidate agent, and a decrease in the level ubiquitin conjugation isindicative of an inhibitory activity for the candidate agent. Asdescribed below, the level of ubiquitination of the p63 protein can bemeasured by determining the actual concentration of protein:ubiquitinconjugates formed; or inferred by detecting some other quality of thesubject protein affected by ubiquitination, including the proteolyticdegradation of the protein. A statistically significant decrease inubiquitination of the p63 protein in the presence of the test compoundis indicative of the test compund being, as appropriately inferred fromthe assay format, an inhibitor of ubiquitin conjugation to p63 and/orubiquitin-mediated degradation of p63.

[0278] In preferred in vitro embodiments of the present assay, theubiquitin-conjugating system comprises a reconstituted protein mixtureof at least semi-purified proteins. With respect to measuringubiquitination, the purified protein mixture can substantially lack anyproteolytic activity which would degrade the p63 substrate proteinand/or components of the ubiquitin conjugating system. For instance, thereconstituted system can be generated to have less than 10% of theproteolytic activity associated with a typical reticulocyte lysate, andpreferably no more than 5%, and most preferably less than 2%.Alternatively, the mixture can be generated to include, either from theonset of ubiquitination or from some point after ubiquitin conjugationof the p63 protein, a ubiquitin-dependent proteolytic activity, such asa purified proteosome complex, that is present in the mixture atmeasured amounts.

[0279] In the subject method, ubiquitin conjugating systems derived frompurified proteins hold a number of significant advantages over celllysate or wheat germ extract based assays (collectively referred tohereinafter as “lysates”). Unlike the reconstituted protein system,without knowledge of particular kinetic parameters for Ub-independantand Ub-dependent degradation of the p63 protein in the lysate,discerning between the two pathways can be extremely difficult.Measuring these parameters, if at all possible, is further made tediousby the fact that cell lysates tend to be inconsistent from batch tobatch, with potentially significant variation between preparations.Evaluation of a potential inhibitor using a lysate system is alsocomplicated in those circumstances where the lysate is charged with mRNAencoding the p63 protein, as such lysates may continue to synthesize theprotein during the assay, and will do so at unpredictable rates.

[0280] Using similar considerations, knowledge of the concentration ofeach component of the ubiquitin conjugation pathway can be required foreach lysate batch, along with the degradative kinetic data, in order todetermine the necessary time course and calculate the sensitivity ofexperiments performed from one lysate preparation to the next.

[0281] Furthermore, the lysate system can be unsatisfactory where thep63 protein itself has a relatively short half-life, especially if dueto degradative processes other than the ubiquitin-mediated pathway towhich an inhibitor is sought. For example, in assays for an inhibitor ofHPV-induced ubiquitination of p53, lysate based systems can be difficultto use, in addition to the reasons set forth above, due to the shorthalf-life of p53 even in extracts which lack HPV proteins. In suchsystems, the ability to measure HPV-mediated ubiquitination of p53 ismade difficult by the already rapid, ongoing degradation of p53presumably occurring by proteolytic processes which are not mediated byany HPV proteins.

[0282] The use of reconstituted protein mixtures allows more carefulcontrol of the reaction conditions in the ubiquitin-conjugating system.Moreover, the system can be derived to favor discovery of inhibitors ofparticular steps of the ubiquitination process. For instance, areconstituted protein assay can be generated which does not facilitatedegradation of the ubiquitinated p63 protein. The level of ubiquitinconjugated p63 can easily be measured directly in such as system, bothin the presence and absence of a candidate agent, thereby enhancing theability to detect a inhibitor of p63 ubiquitination. Alternatively, theUb-conjugating system can be allowed to develop a steady state level ofp63:Ub conjugates in the absence of a proteolytic activity, but thenshifted to a degradative system by addition of purified Ub-dependentproteases. Such degradative systems would be amenable to identifyingdirect inhibitors of ubiquitin-mediated proteolysis of p63.

[0283] The purified protein mixture includes a purified preparation ofthe p63 protein and ubiquitin under conditions which drive theconjugation of the two molecules. For instance, the mixture can includea ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2),and a nucleotide triphosphate (e.g. ATP). Alternatively, the E1 enzyme,the ubiquitin, and the nucleotide triphosphate can be substituted in thesystem with a pre-activated ubiquitin in the form of an E1::Ubconjugate. Likewise, a pre-activated ubiquitin can instead comprise anE2::Ub conjugate which can directly transfer the pre-activated ubiquitinto the p63 protein substrate. Furthermore, the reconstituted mixture canalso be generated to include at least one auxiliary substraterecognition protein (E3) which may be, for example, of cellular or viralorigin. In illustrative embodiments described below, in order togenerate an assay which approximates the ubiquitination of p63 in HPV-16or HPV-18 infected cells, the reconstitutated ubiquitin conjugatingsystem may further include an E6 protein of HPV origin, as well as anE6-associated protein (E6-AP) of cellular origin.

[0284] In one embodiment of the present assay, the products of anon-degradative ubiquitin-conjugating system are separated by gelelectrophoresis, and the level of ubiquitinated p63 protein assessed,using standard electrophesis protocols, by measuring an increase inmolecular weight of the p63 protein that corresponds to the addition ofone or more ubiquitin chains. For example, one or both of the p63protein and ubiquitin can be labeled with a radioisotope such as ³⁵S,¹⁴C, or ³H, and the isotopically labeled protein bands quantified byautoradiographic techniques. Standardization of the assay samples can beaccomplished, for instance, by adding known quantities of labeledproteins which are not themselves subject to ubiquitination ordegradation under the conditions which the assay is performed.Similarly, other means of detecting electrophoretically separatedproteins can be employed to quantify the level of ubiquitination of thep63 protein, including immunoblot analysis using antibodies specific foreither the p63 protein or ubiquitin, or derivatives thereof. Asdescribed below, the antibody can be replaced with another molecule ableto bind one of either the p63 protein or ubiquitin. By way ofillustration, one embodiment of the present assay comprises the use ofbiotinylated ubiquitin in the conjugating system. The biotin label isdetected in a gel during a subsequent detection step by contacting theelectrophoretic products (or a blot thereof) with astreptavidin-conjugated label, such as a streptavidin linkedfluorochrome or enzyme, which can be readily detected by conventionaltechniques. Moreover, where a reconstituted protein mixture is used(rather than a lysate) as the conjugating system, it may be possible tosimply detect the p63 protein and ubiquitin conjugates in the gel bystandard staining protocols, including coomassie blue and silverstaining.

[0285] In another embodiment, an immunoassay or similar binding assay,is used to detect and quantify the level of ubiquitinated p63 proteinproduced in the ubiquitin-conjugating system.

[0286] Many different immunoassay techniques are amenable for such useand can be employed to detect and quantitate the p63 protein:Ubconjugates. For example, the wells of a microtitre plate (or othersuitable solid phase) can be coated with an antibody which specificallybinds one of either the p63 protein or ubiquitin. After incubation ofthe ubiquitin-conjugated system with and without the candidate agent,the products are contacted with the matrix bound antibody, unboundmaterial removed by washing, and ubiquitin conjugates of the p63 proteinspecifically detected. To illustrate, if an antibody which binds the p63protein is used to sequester the protein on the matrix, then adetectable anti-ubiquitin antibody can be used to score for the presenceof ubiquitinated p63 protein on the matrix.

[0287] In similar fashion, epitope-tagged ubiquitin, such as myc-ub (seeEllison et al. (1991) J. Biol. Chem. 266:21150-21157; ubiquitin whichincludes a 10-residue sequence encoding a protein of c-myc) can be usedin conjunction with antibodies to the epitope tag. A major advantage ofusing such an epitope-tagged ubiquitin approach for detecting Ub:proteinconjugates is the ability of an N-terminal tag sequences to inhibitubiquitin-mediated proteolysis of the conjugated p63 protein.

[0288] Other ubiquitin derivatives include detectable labels which donot interfere greatly with the conjugation of ubiquitin to the p63protein. Such detectable lables can include fluorescently-labeled (e.g.FITC) or enzymatically-labeled ubiquitin fusion proteins. Thesederivatives can be produced by chemical cross-linking, or, where thelabel is a protein, by generation of a fusion protein. Several labeledubiquitin derivatives are commercially available.

[0289] Moreover, the p63 protein can be generated as aglutathione-S-transferase (GST) fusion protein. As a practical matter,such GST fusion protein can enable easy purification of the p63 proteinin the preparation of components of the ubiquitin-conjugating system(see, for example, Current Protocols in Molecular Biology, eds. Ausubelet al. (NY: John Wiley & Sons, 1991); Smith et al. (1988) Gene 67:31;and Kaelin et al (1992) Cell 70:351) Moreover, glutathione derivatizedmatrices (e.g. glutathione-sepharose or glutathione-coated microtitreplates) can be used to sequester free and ubiquitinated forms of the p63protein from the ubiguitin-conjugating system, and the level ofubiquitin immobilized can be measured as described. Likewise, where thematrix is generated to bind ubiquitin, the level of sequestered GST-p63protein can be detected using agents which bind to the GST moiety (suchas anti-GST antibodies), or, alternatively, using agents which areenzymatically acted upon by GST to produce detectable products (e.g.1-chloro-2,4-dinitrobenzene; Habig et al. (1974) J Biol Chem 249:7130).Similarly, other fusion proteins involving the p63 protein and anenzymatic activity are contemplated by the present method. For example,fusion proteins containing β-galactosidase or luciferase, to name but afew, can be employed as labels to determine the amount of p63 proteinsequestered on a matrix by virtue of a conjugated ubiquitin chain.

[0290] Moreover, such enzymatic fusion proteins can be used to detectand quantitate ubiquitinated p63 protein in a heterogeneous assay, thatis one which does not require separation of the components of theconjugating system. For example, ubiquitin conjugating systems can begenerated to have a tibiquitin-dependent protease which degrades the p63protein. The enzymatic activity of the fusion protein provides adetectable signal, in the presence of substrate, for measuring the levelof the p63 protein ubiquitination. Similarly, in a non-degradativeconjugating system, ubiquitination of the p63 protein portion of thefusion protein can allosterically influence the enzymatic activityassociated with the fusion the protein and thereby provides a means formonitoring the level of ubiquitin conjugation.

[0291] In binding assay-type detection steps set out above, the choiceof which of either the p63 protein or ubiquitin should be specificallysequestered on the matrix will depend on a number of factors, includingthe relative abundance of both components in the conjugating system. Forinstance, where the reaction conditions of the ubiquitin conjugatingsystem provide ubiquitin at a concentration far in excess of the levelof the p63 protein, (e.g., one order of magnitude or greater)sequestering the ubiquitin and detecting the amount of p63 protein boundwith the ubiquitin can provide less dynamic range to the detection stepof the present method than the converse embodiment of sequestering thep63 protein and detecting ubiquitin conjugates from the total p63protein bound to the matrix. That is, where ubiquitin is provided ingreat excess relative to the p63 protein, the percentage of ubiquitinconjugated p63 protein in the total ubiquitin bound to the matrix can besmall enough that any diminishment in ubiquitination caused by aninhibitor can be made difficult to detect by the fact that, for example,the statistical error of the system (e.g. the noise) can be asignificant portion of the measured change in concentration of bound p63protein. Furthermore, it is clear that manipulating the reactionconditions and reactant concentrations in the ubiquitin-conjugatingsystem can be carried out to provide, at the detection step, greatersensitivity by ensuring that a strong ubiquitinated protein signalexists in the absence of any inhibitor.

[0292] Furthermore, drug screening assays can be generated which do notmeasure ubiquitination per se, but rather detect inhibitory agents onthe basis of their ability to interfere with binding of p63 with anyimmediate upstream or downstream component of the ubiquitin conjugationor proteolysis pathways. Such assays, which are based on disruptingprotein-protein interactions, can be carried out as described above forother p63 interactors.

[0293] In still further embodiments of the present assay, theubiquitin-conjugating system is generated in whole cells, takingadvantage of cell culture techniques to support the subject assay. Forexample, as described below, the ubiquitin-conjugating system (includingthe p63 protein and detection means) can be constituted in a eukaryoticcell culture system, including mammalian and yeast cells. Advantages togenerating the subject assay in an intact cell include the ability todetect inhibitors which are functional in an environment more closelyapproximating that which therapeutic use of the inhibitor would require,including the ability of the agent to gain entry into the cell.Furthermore, certain of the in vivo embodiments of the assay, such asexamples given below, are amenable to high through-put analysis ofcandidate agents.

[0294] The components of the ubiquitin-conjugating system, including thep63 protein, can be endogenous to the cell selected to support theassay. Alternatively, some or all of the components can be derived fromexogenous sources. In any case, the cell is ultimately manipulated afterincubation with a candidate inhibitor in order to facilitate detectionof ubiquitination or ubiquitin-mediated degradation of the p63 protein.As described above for assays performed in reconstituted proteinmixtures or lysate, the effectiveness of a candidate inhibitor can beassessed by measuring direct characteristics of the p63 protein, such asshifts in molecular weight by electrophoretic means or detection in abinding assay. For these embodiments, the cell will typically be lysedat the end of incubation with the candidate agent, and the lysatemanipulated in a detection step in much the same manner as might be thereconstituted protein mixture or lysate.

[0295] Indirect measurement of ubiquitination of the p63 protein canalso be accomplished by detecting a biological activity associated withthe p63 protein that is either attenuated by ubiquitin-conjugation ordestroyed along with the p63 protein by ubiquitin-dependent proteolyticprocesses. As set out above, the use of fusion proteins comprising thep63 protein and an enzymatic activity are representative embodiments ofthe subject assay in which the detection means relies on indirectmeasurement of ubiquitination of the p63 protein by quantitating anassociated enzymatic activity.

[0296] Where the p63 protein has a relatively short half-life due toubiquitin-dependent or independent degradation in the cell, preferredembodiments of the assay either do not require cell lysis, or,alternatively, generate a longer lived detection signal that isindependent of the p63 protein's fate after lysis of the cell. Withrespect to the latter embodiment, the detection means can comprise, forexample, a reporter gene construct which includes a positivetranscriptional regulatory element that binds and is responsive to thep63 protein. For instance, p63 responsive elements can be used toconstruct the reporter gene. These can include a creatine kinaseenhancer, an interleukin-6 promoter, a c-fos promoter, a β-actinpromoter, an hsc70 promoter, a c-jun promoter, a p53 promoter, and aCYC1 hybrid promoter containing a p53/p63-binding sequence. The geneproduct is a detectable label, such as luciferase or β-galactosidase, ora selectable marker, such as an enzyme which confers resistance toantibiotic or other drug, and is produced in the intact cell. The labelcan be measured in a subsequent lysate of the cell. However, the lysisstep is preferably avoided, and providing a step of lysing the cell tomeasure the label will typically only be employed where detection of thelabel cannot be accomplished in whole cells. Such embodiments of thesubject assay are particularly amenable to high through-put analysis inthat proliferation of the cell can provide a simple measure ofinhibition of the ubiquitin-mediated degradation of the p63 protein.

[0297] To illustrate, the plasmid pTKluc described in PCT PublicationWO95/18974 comprises a luciferase gene whose expression is driven by thecore Herpes simplex virus thymidine-kinase (TK) promoter which has beenmodified with either p53 (p53RE/TK), myc (mycRE/TK), or Sp1 (Sp1RE/TK)binding sites. This reporter gene construct is expected to be sensitiveto the level of p63 in the cell. For instance, When the constructlacking any of the modifications to the TK promoter is transfected intomammalian cells, the detectable luciferase activity should be lowbecause this core TK promoter fragment does not contain the upstreamactivating sequences necessary for efficient transcriptional activationof the luciferase gene by p53, and accordingly, should not betransactivated by p63. However transfection with the constructs in whichTK is further modified to contain either 3 or 6 response-elements (RE)for one of p53, myc or Sp 1, the detectable luciferase activity shouldincreases in cells which express appropriate forms of p63. For example,the level of luciferase expression is significantly higher inp53-producing cells (e.g. ML1 cells) transfected with thep53RETK-containing construct than with the TK construct. Likewise,endogenous myc and Sp1 proteins can drive expression of the mycRE/TK andSp1RE/TK constructs. As set out above, it is expected that p63 will bedegraded by the ubiquitin pathway. However, Sp1 is not known to bedegraded by any ubiquitin-mediated pathway, and the SPIRE/TK constructcan therefore be used as a control in the present assays. Thus, in thepresence of an agent which inhibits ubiquitin-mediated degradation ofp63 in a cell harboring a p53RE/TK construct, the level of luciferaseactivity would increase relative to that in the cell not treated withthe candidate agent.

[0298] 4.8 Diagnostic and Prognostic Assays

[0299] The present methods provide means for determining if a subject isat risk for developing a disease or condition associated with disordermarked by an aberrant p63 activity, e.g., an aberrant level of p63protein or particular isoform thereof. As set forth below, diseases orconditions that can be caused by an abnormal p63 level or catalyticactivity include diseases or conditions caused by or contributed to byan abnormal amount of a target peptide of p63.

[0300] According to a diagnostic method of the present invention, lossof the wild-type p63 is detected. This loss may be due to eitherdeletional and/or point mutational events. If only a single p63 alleleis mutated, an early neoplastic state is indicated. However, if bothalleles are mutated then a late neoplastic state is indicated. The p63allele which is not deleted (i.e., that on the sister chromosome to thechromosome carrying the deletion) can be screened for point mutations,such as missense, and frameshift mutations. These mutations could leadto non-functional p63 gene products. In addition, the point mutationalevents may occur in the regulatory regions, such as in the promoter ofthe p63 gene, could lead to a loss or dimunition of expression of thep63 mRNA.

[0301] In order to detect the loss of the p63 wild-type gene in tissue,it is helpful to isolate the tissue from the surrounding normal tissues.Means for enriching tumor preparations are known in the art, e.g.,cytometry. Detection of point mutations may be accomplished by molecularcloning of the p63 allele (or alleles) present in tumor tissue.Alternatively, the polymerase chain reaction can be used to amplify p63gene sequences directly from a genomic DNA preparation. The DNA sequenceof the amplified sequences can be determined.

[0302] Specific deletions of the p63 gene can also be detected. Forexample, restriction fragment length polymorphism (RFLP) probes for thep63 genes may also be used to score the loss of a p63 allele. Loss ofthe wild-type p63 genes may also be detected on the basis of the loss ofthe wild type expression products of p63. Such expression productsinclude the mRNA as well as the p63 protein product itself.

[0303] In one embodiment, the diagnostic method comprises determiningwhether a subject has an abnormal mRNA and/or protein level of p63, suchas by Northern blot analysis, reverse transcription—polymerase chainreaction (RT-PCR), in situ hybridization, immunoprecipitation, Westernblot hybridization, or immunohistochemistry. According to the method,cells are obtained from a subject and the p63 protein or mRNA level isdetermined and compared to the level of p63 protein or mRNA level in ahealthy subject. An abnormal level of p63 polypeptide or mRNA level islikely to be indicative of an aberrant p63 activity.

[0304] In another embodiment, the diagnostic method comprises measuringat least one activity of p63. For example, the ability to inducetransactivation of target genes, e.g. genes involved in cell cyclearrest. Comparison of the results obtained with results from similaranalysis performed on p63 proteins from healthy subjects will beindicative of whether a subject has an abnormal p63 activity.

[0305] In preferred embodiments, the methods for determining whether asubject is at risk for developing a disease, such as a predisposition todevelop tumors, associated with an aberrant p63 activity ischaracterized as comprising detecting, in a sample of cells from thesubject, the presence or absence of a genetic lesion characterized by atleast one of (i) an alteration affecting the integrity of a geneencoding a p63 polypeptide, or (ii) the mis-expression of the p63 gene.To illustrate, such genetic lesions can be detected by ascertaining theexistence of at least one of (i) a deletion of one or more nucleotidesfrom a p63 gene, (ii) an addition of one or more nucleotides to a p63gene, (iii) a substitution of one or more nucleotides of a p63 gene,(iv) a gross chromosomal rearrangement of a p63 gene, (v) a grossalteration in the level of a messenger RNA transcript of a p63 gene,(vii) aberrant modification of an p63 gene, such as of the methylationpattern of the genomic DNA, (vii) the presence of a non-wild typesplicing pattern of a messenger RNA transcript of a p63 gene, (viii) anon-wild type level of a p63 polypeptide, (ix) allelic loss of a p63gene, and/or (x) inappropriate post-translational modification of a p63polypeptide. As set out below, the present invention provides a largenumber of assay techniques for detecting lesions in a p63 gene. Thesemethods include, but are not limited to, methods involving sequenceanalysis, Southern blot hybridization, restriction enzyme site mapping,and methods involving detection of absence of nucleotide pairing betweenthe nucleic acid to be analyzed and a probe. These and other methods arefurther described infra.

[0306] Specific diseases or disorders, e.g., genetic diseases ordisorders, are associated with specific allelic variants of polymorphicregions of certain genes, which do not necessarily encode a mutatedprotein. Thus, the presence of a specific allelic variant of apolymorphic region of a gene in a subject can render the subjectsusceptible to developing a specific disease or disorder. Polymorphicregions in genes, e.g, p63 genes, can be identified, by determining thenucleotide sequence of genes in populations of individuals. If apolymorphic region is identified, then the link with a specific diseasecan be determined by studying specific populations of individuals, e.g,individuals which developed a specific disease, such as hypertension. Apolymorphic region can be located in any region of a gene, e.g., exons,in coding or non coding regions of exons, introns, and promoter region.

[0307] In an exemplary embodiment, there is provided a nucleic acidcomposition comprising a nucleic acid probe including a region ofnucleotide sequence which is capable of hybridizing to a sense orantisense sequence of a p63 gene or naturally occurring mutants thereof,or 5′ or 3′ flanking sequences or intronic sequences naturallyassociated with the subject p63 genes or naturally occurring mutantsthereof. The nucleic acid of a cell is rendered accessible forhybridization, the probe is contacted with the nucleic acid of thesample, and the hybridization of the probe to the sample nucleic acid isdetected. Such techniques can be used to detect lesions or allelicvariants at either the genomic or mRNA level, including deletions,substitutions, etc., as well as to determine mRNA transcript levels.

[0308] A preferred detection method is allele specific hybridizationusing probes overlapping the mutation or polymorphic site and havingabout 5, 10, 20, 25, or 30 nucleotides around the mutation orpolymorphic region. In a preferred embodiment of the invention, severalprobes capable of hybridizing specifically to allelic variants areattached to a solid phase support, e.g., a “chip”. Oligonucleotides canbe bound to a solid support by a variety of processes, includinglithography. For example a chip can hold up to 250,000 oligonucleotides(GeneChip, Affymetrix). Mutation detection analysis using these chipscomprising oligonucleotides, also termed “DNA probe arrays” is describede.g., in Cronin et al. (1996) Human Mutation 7:244. In one embodiment, achip comprises all the allelic variants of at least one polymorphicregion of a gene. The solid phase support is then contacted with a testnucleic acid and hybridization to the specific probes is detected.Accordingly, the identity of numerous allelic variants of one or moregenes can be identified in a simple hybridization experiment.

[0309] In certain embodiments, detection of the lesion comprisesutilizing the probe/primer in a polymerase chain reaction (PCR) (see,e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACEPCR, or, alternatively, in a ligase chain reaction (LCR) (see, e.g.,Landegran et al (1988) Science 241:1077-1080; and Nakazawa et al (1994)PNAS 91:360-364), the latter of which can be particularly useful fordetecting point mutations in the p63 gene (see Abravaya et al. (1995)Nuc Acid Res 23:675-682). In a merely illustrative embodiment, themethod includes the steps of (i) collecting a sample of cells from apatient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) fromthe cells of the sample, (iii) contacting the nucleic acid sample withone or more primers which specifically hybridize to a p63 gene underconditions such that hybridization and amplification of the p63 gene (ifpresent) occurs, and (iv) detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

[0310] Alternative amplification methods include: self sustainedsequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad.Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-BetaReplicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill inthe art. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

[0311] In a preferred embodiment of the subject assay, mutations in, orallelic variants, of a p63 gene from a sample cell are identified byalterations in restriction enzyme cleavage patterns. For example, sampleand control DNA is isolated, amplified (optionally), digested with oneor more restriction endonucleases, and fragment length sizes aredetermined by gel electrophoresis. Moreover, the use of sequencespecific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

[0312] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the p63 geneand detect mutations by comparing the sequence of the sample p63 withthe corresponding wild-type (control) sequence. Exemplary sequencingreactions include those based on techniques developed by Maxim andGilbert (Proc. Natl. Acad Sci USA (1977) 74:560) or Sanger (Sanger et al(1977) Proc. Nat. Acad Sci 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures may be utilized whenperforming the subject assays (Biotechniques (1995) 19:448), includingsequencing by mass spectrometry (see, for example PCT publication WO94/16101; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin etal. (1993) Appl Biochem Biotechnol 38:147-159). It will be evident toone skilled in the art that, for certain embodiments, the occurrence ofonly one, two or three of the nucleic acid bases need be determined inthe sequencing reaction. For instance, A-track or the like, e.g., whereonly one nucleic acid is detected, can be carried out.

[0313] In a further embodiment, protection from cleavage agents (such asa nuclease, hydroxylamine or osmium tetroxide and with piperidine) canbe used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labelled) RNA or DNA containing the wild-type p63sequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to base pair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digest the mismatched regions.In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al (1988) Proc. Natl. Acad Sci USA 85:4397; Saleeba et al.(1992) Methods Enzymod. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

[0314] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in p63 cDNAs obtainedfrom samples of cells. For example, the mutY enzyme of E. coli cleaves Aat G/A mismatches and the thymidine DNA glycosylase from HeLa cellscleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on ap63 sequence, e.g., a wild-type p63 sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039.

[0315] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations or the identity of the allelicvariant of a polymorphic region in p63 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad Sci USA 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control p63nucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0316] In yet another embodiment the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing agent gradient to identify differences inthe mobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

[0317] Examples of other techniques for detecting point mutations or theidentity of the allelic variant of a polymorphic region include, but arenot limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl. Acad. Sci USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labelledtarget DNA.

[0318] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation or polymorphic region of interestin the center of the molecule (so that amplification depends ondifferential hybridization) (Gibbs et al (1989) Nucleic Acids Res.17:2437-2448) or at the extreme 3′ end of one primer where, underappropriate conditions, mismatch can prevent, or reduce polymeraseextension (Prossner (1993) Tibtech 11:238. In addition it may bedesirable to introduce a novel restriction site in the region of themutation to create cleavage-based detection (Gasparini et al. (1992)Mol. Cell Probes 6:1). It is anticipated that in certain embodimentsamplification may also be performed using Taq ligase for amplification(Barany (1991) Proc. Natl. Acad Sci USA 88:189). In such cases, ligationwill occur only if there is a perfect match at the 3′ end of the 5′sequence making it possible to detect the presence of a known mutationat a specific site by looking for the presence or absence ofamplification.

[0319] In another embodiment, identification of the allelic variant iscarried out using an oligonucleotide ligation assay (OLA), as described,e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science241:1077-1080 (1988). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g,. biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc.Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

[0320] Several techniques based on this OLA method have been developedand can be used to detect specific allelic variants of a polymorphicregion of an SR-BI gene. For example, U.S. Pat. No. 5,593,826 disclosesan OLA using an oligonucleotide having 3′-amino group and a5′-phosphorylated oligonucleotide to form a conjugate having aphosphoramidate linkage. In another variation of OLA described in To beet al. ((1996)Nucleic Acids Res 24: 3728), OLA combined with PCR permitstyping of two alleles in a single microtiter well. By marking each ofthe allele-specific primers with a unique hapten, i.e. digoxigenin andfluorescein, each OLA reaction can be detected by using hapten specificantibodies that are labeled with different enzyme reporters, alkalinephosphatase or horseradish peroxidase. This system permits the detectionof the two alleles using a high throughput format that leads to theproduction of two different colors.

[0321] The invention further provides methods for detecting singlenucleotide polymorphisms in an p63 gene. Because single nucleotidepolymorphisms constitute sites of variation flanked by regions ofinvariant sequence, their analysis requires no more than thedetermination of the identity of the single nucleotide present at thesite of variation and it is unnecessary to determine a complete genesequence for each patient. Several methods have been developed tofacilitate the analysis of such single nucleotide polymorphisms.

[0322] In one embodiment, the single base polymorphism can be detectedby using a specialized exonuclease-resistant nucleotide, as disclosed,e.g., in Mundy, C. R. (U.S. Pat. No. 4,656,127). According to themethod, a primer complementary to the allelic sequence immediately 3′ tothe polymorphic site is permitted to hybridize to a target moleculeobtained from a particular animal or human. If the polymorphic site onthe target molecule contains a nucleotide that is complementary to theparticular exonuclease-resistant nucleotide derivative present, thenthat derivative will be incorporated onto the end of the hybridizedprimer. Such incorporation renders the primer resistant to exonuclease,and thereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

[0323] In another embodiment of the invention, a solution-based methodis used for determining the identity of the nucleotide of a polymorphicsite. Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No.WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primeris employed that is complementary to allelic sequences immediately 3′ toa polymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

[0324] An alternative method, known as Genetic Bit Analysis or GBA ™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

[0325] Several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA ™ in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer.J. Hum. Genet. 52:46-59 (1993)).

[0326] For mutations that produce premature termination of proteintranslation, the protein truncation test (PTT) offers an efficientdiagnostic approach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21;van der Luijt, et. al., (1994) Genomics 20:1-12). For PTT, RNA isinitially isolated from available tissue and reverse-transcribed, andthe segment of interest is amplified by PCR. The products of reversetranscription PCR are then used as a template for nested PCRamplification with a primer that contains an RNA polymerase promoter anda sequence for initiating eukaryotic translation. After amplification ofthe region of interest, the unique motifs incorporated into the primerpermit sequential in vitro transcription and translation of the PCRproducts. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresisof translation products, the appearance of truncated polypeptidessignals the presence of a mutation that causes premature termination oftranslation. In a variation of this technique, DNA (as opposed to RNA)is used as a PCR template when the target region of interest is derivedfrom a single exon.

[0327] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid, primer set; and/or antibody reagent described herein,which may be conveniently used, e.g., in clinical settings to diagnosepatients exhibiting symptoms or family history of a disease or illnessinvolving a p63 polypeptide.

[0328] Any cell type or tissue may be utilized in the diagnosticsdescribed below. In a preferred embodiment a bodily fluid, e.g., blood,is obtained from the subject to determine the presence of a mutation orthe identity of the allelic variant of a polymorphic region of an p63gene. A bodily fluid, e.g, blood, can be obtained by known techniques(e.g. venipuncture). Alternatively, nucleic acid tests can be performedon dry samples (e.g. hair or skin). For prenatal diagnosis, fetalnucleic acid samples can be obtained from maternal blood as described inInternational Patent Application No. WO91/07660 to Bianchi.Alternatively, amniocytes or chorionic villi may be obtained forperforming prenatal testing.

[0329] When using RNA or protein to determine the presence of a mutationor of a specific allelic variant of a polymorphic region of a p63 gene,the cells or tissues that may be utilized must express the p63 gene.Preferred cells for use in these methods include megakaryocytes, whichhave been shown to express the 3 zinc finger proteins of the invention(see Examples). Alternative cells or tissues that can be used, can beidentified by determining the expression pattern of the specific p63gene in a subject, such as by Northern blot analysis.

[0330] Diagnostic procedures may also be performed in situ directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents may be used as probes and/or primersfor such in situ procedures (see, for example, Nuovo, G. J., 1992, PCRin situ hybridization: protocols and applications, Raven Press, NY).

[0331] In addition to methods which focus primarily on the detection ofone nucleic acid sequence, profiles may also be assessed in suchdetection schemes. Fingerprint profiles may be generated, for example,by utilizing a differential display procedure, Northern analysis and/orRT-PCR.

[0332] Antibodies directed against wild type or mutant p63 polypeptidesor allelic variant thereof, which are discussed above, may also be usedin disease diagnostics and prognostics. Such diagnostic methods, may beused to detect abnormalities in the level of p63 polypeptide expression,or abnormalities in the structure and/or tissue, cellular, orsubcellular location of a p63 polypeptide. Structural differences mayinclude, for example, differences in the size, electronegativity, orantigenicity of the mutant p63 polypeptide relative to the normal p63polypeptide. Protein from the tissue or cell type to be analyzed mayeasily be detected or isolated using techniques which are well known toone of skill in the art, including but not limited to western blotanalysis. For a detailed explanation of methods for carrying out Westernblot analysis, see Sambrook et al, 1989, supra, at Chapter 18. Theprotein detection and isolation methods employed herein may also be suchas those described in Harlow and Lane, for example, (Harlow, E. andLane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which is incorporatedherein by reference in its entirety.

[0333] This can be accomplished, for example, by immunofluorescencetechniques employing a fluorescently labeled antibody (see below)coupled with light microscopic, flow cytometric, or fluorimetricdetection. The antibodies (or fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence or immunoelectron microscopy, for in situ detectionof p63 polypeptides. In situ detection may be accomplished by removing ahistological specimen from a patient, and applying thereto a labeledantibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the p63 polypeptide, but also itsdistribution in the examined tissue. Using the present invention, one ofordinary skill will readily perceive that any of a wide variety ofhistological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

[0334] Often a solid phase support or carrier is used as a supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

[0335] One means for labeling an anti-p63 polypeptide specific antibodyis via linkage to an enzyme and use in an enzyme immunoassay (EIA)(Voller, “The Enzyme Linked Immunosorbent Assay (ELISA)”, DiagnosticHorizons 2:1-7, 1978, Microbiological Associates Quarterly Publication,Walkersville, Md.; Voller, et al., J. Clin. Pathol. 31:507-520 (1978);Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, (ed.) EnzymeImmunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa, et al., (eds.)Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

[0336] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect fingerprintgene wild type or mutant peptides through the use of a radioimmunoassay(RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques, The EndocrineSociety, March, 1986, which is incorporated by reference herein). Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography.

[0337] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0338] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0339] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0340] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0341] Moreover, it will be understood that any of the above methods fordetecting alterations in a gene or gene product or polymorphic variantscan be used to monitor the course of treatment or therapy.

[0342] 4.9 Methods of Treating Diseases

[0343] In general, the invention provides methods for treating orpreventing a disease caused by or contributed to by aberrant expressionor activity of a p63 gene product, comprising administering to thesubject an effective amount of a pharmaceutical composition comprising acompound which is capable of modulating a p63 activity, such that thedisease is treated or prevented in the subject. Among the approacheswhich may be used to ameliorate disease symptoms involving an aberrantp63 activity are, for example, gene therapy, antisense, ribozyme, andtriple helix molecules described above. Other suitable compounds includethe compounds identified in the drug screening assays above, as well asthe various antagonists and agonist forms of the protein.

[0344] In a preferred embodiment, the compounds of the present inventionare useful for regulating tumorigensis. In fact, based on thesignificant nucleotide and amino acid sequence homology with p53, p63 isa logical target for cancer therapy. The mutation spectrum of p53provide clues to the critical functional regions of the gene, that, whenmutated, contribute to the carcinogenic process. Since about 80% of themissense mutations are in the sequence-specific DNA binding midregion ofthe protein, investigators have focused on the transcriptiontransactivator function of p53. However, these missense mutations andthe resultant amino acid substitutions can cause aberrant proteinconformations that also may alter other functional domains, includingthose in the carboxyl-terminus of the p53 protein. This positivelycharged region contains the putative major nuclear localization signal(amino acids 316-325), the oligomerization domain (amino acids 319-360),and a DNA damage-binding domain (amino acids 318-393). p53sequence-specific DNA binding and transcriptional transactivation canalso be modulated by post-translational mechanisms, including serinephosphorylation and the redox regulation of the cysteine residuesresponsible for binding zinc to p53. The structure-function relationshiprevealed by the analysis of the p53 mutation spectrum, provides us withstrategies for the development of rational cancer therapy; particularlybecause of the considerable sequence identity between p53 and p63. Asdiscussed above, it has been observed that the sequence identity betweenp63 and p73 alpha form is about 57.4% identity and the p63 and p73 betaform shows about 69.7% identity at the protein level. p63 alpha form andp53 exhibit 43.8% identity at the protein level.

[0345] In one embodiment, this invention is directed to the developmentof drugs to mimic the cell regulator function of p63. Strategies toscreen potential drugs are suggested by the development of assaysreflecting the biologic functions of the p63 protein: its binding tospecific DNA sequences, its function as a transcription factor, itsfunction as an inducer of apoptosis, and its ability to form complexeswith cellular or viral oncoproteins. For instance, in one embodiment,apoptosis, the cell death pathway may be enhanced by anticancertherapies. Cells exposed to agents that produce DNA damage, such asdouble-strand breaks, may use the p53/p63-mediated pathway to induceapoptosis. It is known in the art that certain cell growth factors actas survival factors of cancer cells, therefore, reduction of thesefactors would produce apoptosis, e.g., the use of anti-EGF-recptormonoclonal antibodies, which block the EGF mediated growth signalcascade, have been shown to act synergistically with anti-cancer drugs.Thus, modulating the survival factors, survival factor pathways, theircellular receptors and inhibitors provide novel methods of inhibitingtumorigeneisis. It is possible that p63 may mediate apoptosis by bothtranscriptional transactivation of genes that enhance apoptosis andtranscription transrepression of genes that inhibit apoptosis. Thesegenes their encoded proteins may also be targets for therapeuticstrategies.

[0346] It is known in the art that certain DNA viruses have oncoproteinsthat that bind to p53 and inactivate its functions, it is likely thatthese would also inactivate the functions of the closely analogous p63.The E6 protein of the oncogenic strains of the human papillomavirusbinds to p53 via E6-AP, a specific ubiquitin protein ligase and enhancesthe digestion of p53, and possible th edigestion of p63. Drugs thatinhibit either the formation of this protein complex or the digestion ofeither p53 or p63, might have a therapeutic benefit in tumors associatedwith human papillomavirus infections, such as cervical, penile, andrectal carcinomas.

[0347] p63 has been mapped to chromosome 3, i.e., 3q27. Deletions ofregions of chromosome 3 have been implicated in lung, uterine, breast,testicular, and ovarian cancers, von Hippel-Lindau and 3p deletionsyndrome, renal cell and nasopharyngeal carcinomas, mesotheliomas, andvarious haematological malignancies. In particular, chromosomal breaksat 3q27 have been implicated in intermediate grade non-Hodgkin'slymphomas (NHL). The pathologic heterogeneity of NHL is reflected at thecellular level by the various pathways underlying NHL pathogenesis. Inparticular, two main categories of genetic lesions, activation ofdominantly acting oncogenes and deletion of tumor-suppression genes areknown in the art as contributing to lymphonogenesis. In particular,oncogene activation in NHL most commonly occurs through non-randomchromosomal translocations.

[0348] In many instances, these translocations involve reciprocalexchanges between an antigen receptor locus (the immunoglobulin loci orthe TCR in B- and T-cell malignancies, respectively) and variousprotooncogene loci. Once the protooncogne is juxtaposed to an antigenreceptor locus, its expression becomes regulated by immunoglogulin TCRpromoters and enhancers. The resulting transcriptional deregulation maybe one factor causing activation of the translocated protooncogene.Accordingly, in one embodiment it is contemplated that overexpression ofthe p63-B forms may be implicated in NHL. In yet another embodiment, theinvention contemplates the use of a combination of strategies, forexample, a low dose of a DNA damaging agent to arrest normal cells in G₁of the cell cycle and a delayed dose of an antimitotic agent to targetthe mutant p63 tumor cells that continue to progress into the S phase,G₂, and mitosis.

[0349] As observed in the case of p53, it is likely that tumor derivedp63 mutations will target amino acid residues that are important for thestructural integrity of the core domain of p63. Failure of muatntproteins to bind to DNA has been attributed to the structural defects inthe proteins such as structural rearrangements, local unfolding of thestructure, or denaturation of the core domain. Therefore, mutant p63 canhave altered sequence specific DNA-binding and function as atranscription factor either by inhibiting its transactivator activity orby changing its specificity of DNA binding and the repertoire of genesthat are transcriptionally transactivated. Although, it would seemdifficult to reverse mutant conformations to the wild type, numerousstrategies have been considered. First, certain like p53, certain p63mutant proteins are believed to have temperature sensitive phenotypes,including increased transcription-transcativator and growth inhibitionactivities at the lower permissive temperature when compared to thenon-permissive higher temperature. Second, certain peptide drugs canlater the conformation of mutant p63 in cells. Third, certain p63mutants can still form tetramers and cooperate with transfectedwild-type p63 in the transcriptional tranactivation of reporter geneconstructs. It would appear that p63 missense mutants most likely toassume a wild-type protein conformation apear to be those with asubstituted amino acid in the sequence specific DNA binding site.Mutations resulting in amino acid substitutions in the interior of thep63 protein may be a thermodynamically less stable folded structure andrequire other strategies. Tumors having these interior p63 mutationsalso bind to cellular proteins which could lead to either dominantnegative or gain of oncogenic activities. Therefore, strategies such astargeting the mutant gene by triple Dna helox and antisense approachescould result in diminishing these activities and have a therapeuticbenefit.

[0350] The efficacy of p53 gene therapy in human cancer cells has beendemonstrated in vitro by Lee et al., Cancer Metastasis Rev., 14:149-61(1995), the efficacy as xenografts in athymic nude mice has beendemonstrated by Lesoon-Wood et al., Hum. Gene Ther. 6:395-405 (1995);Clayman et al., Cancer Res. 55:1-12 (1995); and Liu et al., Cancer Res.55:3117-22 (1995). The p53 gene, i.e., at p53 complementary DNAexpression vector was successfully transferred any transfection orinfection using either a replication defective retroviral or anadenoviral vector and tumor cell growth was inhibited. In fact, thesucces of these expression vectors has led to the approval of phase Iprotocols in humans. In yet another embodiment, the use of the p53complementary DNA expression vectors in gene therapy protocols iscontemplated.

[0351] 4.9.1. Tumor Vaccines

[0352] The treatment of cancer with tumor vaccines has been a goal ofphysicians and scientists ever since effective immunization againstinfectious disease with vaccines was developed. In the past, major tumorantigens had not been molecularly characterized. Recent advances are,however, beginning to define potential molecular targets and strategiesand this had evolved with the principle that T-cell mediated responsesare a key target for approaches to cancer immunization. In addition,these antigens are not truly foreign and tumor antigens fit more with aself/altered self paradigm, compared to a non-self paradigm for antigensrecognized in infectious diseases. Antigens that have been used in theart include the glycolipids and glycoproteins e.g. gangliosides, thedevelopmental antigens, e.g., MAGE, tyrosinase, melan-A and gp75, andmutant oncogene products, e.g., p53, ras, and HER-2/neu. Vaccinepossibilities include purified proteins and glycolipids, peptides, cDNAexpressed in various vectors, and a range of immune adjuvants.

[0353] Yet another aspect of the present invention relates to themodification of tumor cells, and/or the immune response to tumor cellsin a patient by administering a vaccine to enhance the anti-tumor immuneresponse in a host. The present invention provides, for examples, tumorvaccines based on administration of expression vectors encoding a mutanttumor suppressor gene, e.g., p53, p73, or p63, or portions thereof, orimmunogenic preparations of polypeptides.

[0354] In general, it is noted that malignant transformation of cells iscommonly associated with phenotypic changes. Such changes can includeloss, gain, or alteration in the level of expression of certainproteins. It has been observed that in some situations the immune systemmay be capable of recognizing a tumor as foreign and, as such, mountingan immune response against the tumor (Kripke, M., Adv. Cancer Res. 34,69-75 (1981)). This hypothesis is based in part on the existence ofphenotypic differences between tumor cells and normal cells, which issupported by the identification of tumor associated antigens (TAAs)(Schreiber, H., et al. Ann. Rev. Immunol. 6, 465483 (1988)). TAAs arethought to distinguish a transformed cell from its normal counterpart.For example, three genes encoding TAAs expressed in melanoma cells,MAGE-1, MAGE-2 and MAGE-3, have recently been cloned (van der Bruggen,P., et al. Science 254, 1643-1647 (1991)). That tumor cells undercertain circumstances can be recognized as foreign is also supported bythe existence of T cells which can recognize and respond to tumorassociated antigens presented by MHC molecules. Such TAA-specific Tlymphocytes have been demonstrated to be present in the immunerepertoire and are capable of recognizing and stimulating an immuneresponse against tumor cells when properly stimulated in vitro(Rosenberg, S. A., et al. Science 233, 1318-1321 (1986); Rosenberg, S.A. and Lotze, M. T. Ann. Rev. Immunol. 4, 681-709 (1986)). In the caseof melanoma cells both the tyrosinase gene (Brichard, V., et al. J. Exp.Med. 178:489 (1993)) and the Melan-A gene (Coulie et al. J. Exp. Med.180:35)) have been identified as genes coding for antigens recognized byautologous CTL on melanoma cells.

[0355] Induction of T lymphocytes is a critical initial step in a host'simmune response. Activation of T cells results in cytokine production, Tcell proliferation, and generation of T cell-mediated effectorfunctions. T cell activation requires an antigen-specific signal, oftencalled a primary activation signal, which results from stimulation of aclonally-distributed T cell receptor (TcR) present on the surface of theT cell. This antigen-specific signal is usually in the form of anantigenic peptide bound either to a major histocompatibility complex(MHC) class I protein or an MHC class II protein present on the surfaceof an antigen presenting cell (APC). CD4+, helper T cells recognizepeptides associated with class II molecules which are found on a limitednumber of cell types, primarily B cells, monocytes/macrophages anddendritic cells. In most cases class II molecules present peptidesderived from proteins taken up from the extracellular environment. Incontrast, CD8+, cytotoxic T cells (CTL) recognize peptides associatedwith class I molecules. Class I molecules are found on almost all celltypes and, in most cases, present peptides derived from endogenouslysynthesized proteins. For a review see Germain, R., Nature 322, 687-691(1986).

[0356] The importance of T cells in tumor immunity has severalimplications which are important in the development of anti-tumorvaccines. Since antigens are processed and presented before they arerecognized by T cells, they may be derived from any protein of the tumorcell, whether extracellular or intracellular. In addition, the primaryamino acid sequence of the antigen is more important than thethree-dimensional structure of the antigen. Tumor vaccine strategies mayuse the tumor cell itself as a source of antigen, or may be designed toenhance responses against specific gene products. (Pardoll, D. 1993.Annals of the New York Academy of sciences 690:301).

[0357] The present invention provides for various tumor vaccinationmethods and reagents which can be used to elicit an anti-tumor responseagainst transformed cells which express/display a mutant p63, p53, orp73, or which have been engineered to present an antigen of a mutantp63, p53, or p73. In general, the tumor vaccine strategies of thepresent invention fall into two categorie: (1) strategies that use thetumor cell itself as a source of tumor antigen, and (2) antigen-specificvaccine strategies that are designed to generate immune responsesagainst specific antigens of mutant p63, p53, or p73s.

[0358] In general, a p63, p53, or p73 vaccine polypeptide will includeat least a portion of the p63, p53, or p73 polypeptide including a siteof mutation which, when occurring in the full-length protein, results inloss of its biological activity. Where the p63, p53, or p73 tumorvaccine comprises a sufficient portion of a mutant p63, p53, or p73protein, the p63, p53, or p73 protein can be further mutated to renderthe vaccine polypeptide biologically inactive.

[0359] In one embodiment, a tumor cell which otherwise does not expressa mutant p63, p53, or p73 can be rendered immunogenic as a target forCTL recognition by association of a p63, p53, or p73 vaccinepolypeptide. For example, this can be accomplished by the use of genetransfer vectors. Such gene transfer vectors may be administered in anybiologically effective carrier, e.g. any formulation or compositioncapable of effectively delivering the p63, p53, or p73 vaccine gene tocells in vivo. Alternatively, cells from the patient or other hostorganism can be transfected with the tumor vaccine construct ex vivo,allowed to express the p63, p53, or p73 protein, and, preferably afterinactivation by radiation or the like, administered to an individual. Inparticular, viral vectors represent an attractive method for delivery oftumor vaccine antigens because viral proteins are expressed de novo ininfected cells, are degraded within the cytosol, and are transported tothe endoplasmic reticulum where the degraded peptide products associatewith MHC class I molecules before display on the cell surface (Spooneret al. (1995) Gene Therapy 2:173).

[0360] Approaches include insertion of the subject gene into viralvectors including recombinant retroviruses, adenovirus, adeno-associatedvirus, vaccinia virus, and herpes simplex virus-1, or plasmids. Viralvectors transfect cells directly; plasmid DNA can be delivered with thehelp of, for example, cationic liposomes (lipofectin) or derivatized(e.g. antibody conjugated), polylysine conjugates, gramacidin S,artificial viral envelopes or other such intracellular carriers, as wellas direct injection of the gene construct or CaPO₄ precipitation carriedout in vivo. It will be appreciated that because transduction ofappropriate target cells represents the critical first step in genetransfer, choice of the particular gene delivery system will depend onsuch factors as the phenotype of the intended target and the route ofadministration, e.g. locally or systemically.

[0361] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed to cause expression of asubject p63, p53, or p73 polypeptide in the tissue of an animal in orderto ellicit a cellular immune response. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the vaccine gene by thetargeted cell. Exemplary gene delivery systems of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes.

[0362] In another embodiment the mutant p63, p53, or p73 peptides of thepresent invention may be directly delivered to the patient. Althoughsuch expression constructs as exemplified above have been shown to be anefficient means by which to obtain expression of peptides in the contextof class I molecules, vaccination with isolated peptides has also beenshown to result in class I expression of the peptides in some cases. Forexample, the use of synthetic peptide fragments containing CTL epitopeswhich are presented by class I molecules has been shown to be aneffective vaccine against infection with lymphocytic choriomeningitisvirus (Schultz et al. 1991. Proc. Natl. Acad. Sci. USA 88:2283) orsendai virus (Kast et al. 1991. Proc Natl Acad Sci. 88:2283).Subcutaneous administration of a CTL epitope has also been found torender mice resistant to challenge with human papillomavirus16-transformed tumor cells (Feltkamp et al. (1993) Eur. J. Immunol.23:2242-2249). It is contemplated that such peptides may be presented inthe context of tumor cell class I antigens or by other, host-derivedclass I bearing cells (Huang et al. 1994. Science 264:961).

[0363] The mutant p63, p53, or p73 proteins, and portions thereof, maybe used in the preparation of vaccines prepared by known techniques(c.f., U.S. Pat. Nos. 4,565,697; 4,528,217 and 4,575,495). p63, p53, orp73 polypeptides displaying antigenic regions capable of elicitingprotective immune response are selected and incorporated in anappropriate carrier. Alternatively, an antitumor antigenic portion of ap63, p53, or p73 protein may be incorporated into a larger protein byexpression of fused proteins.

[0364] The p63, p53, or p73 antitumor vaccines above may be administeredin any conventional manner, including oranasally, subcutaneously,intraperitoneally or intramuscularly. The vaccine may further comprise,as discussed infra, an adjuvant in order to increase the immunogenicityof the vaccine preparation.

[0365] In some cases it may be advantageous to couple the p63, p53, orp73 polypeptide vaccine to a carrier, in particular a macromolecularcarrier. The carrier can be a polymer to which the p63, p53, or p73polypeptide is bound by hydrophobic non-covalent inneraction, such as aplastic, e.g., polystyrene, or a polymer to which the polypeptide iscovalently bound, such as a polysaccharide, or a polypeptide, e.g.,bovine serum albumin, ovalbumin or keyhole limpet hemocyanin. Thecarrier should preferably be non-toxic and non-allergenic. The p63, p53,or p73 polypeptide may be multivalently coupled to the macromolecularcarrier as this provides an increased immunogenicity of the vaccinepreparation. It is also contemplated that the p63, p53, or p73polypeptide may be presented in multivalent form by polymerizing thepolypeptide with itself.

[0366] In addition, the vaccine formulations may also contain one ormore stabilizer, exemplary being carbohydrates such as sorbitol,mannitol, starch, sucrose, dextrin, and glucose, proteins such asalbumin or casein, and buffers such as alkaline metal phosphate and thelike.

[0367] The inclusion of CD4+ epitopes in the tumor vaccine in order tofurther enhance an anti-tumor response is also within the scope of theinvention.

[0368] In other embodiments, the tumor cell itself can be used as thesource of antitumor p63, p53, or p73 antigens. See, for review, Pardoll,D. 1993. Annals of the New York Academy of Sciences 690:301. Forexample, cells which have been identified through phenotyping asexpressing a mutant p63, p53, or p73 can be used to generate a CTLresponse against a tumor. For example, tumor-infiltrating lymphocytes(TILs) may be derived from tumor biopsies which have such a phenotype.Following such protocols as described by Hom et al. (1991) JImmunotherap 10: 153, TILs can be isolated from tumor specimens andgrown in the presence of interleukin-2 in order to generate oligoclonalpopulations of activated T-lymphocytes that are cytolytic to the tumorcells expressing the mutant p63, p53, or p73.

[0369] In other embodiments, whole cell vaccines can be used to treatcancer patients. Such vaccines can include, for example, irradiatedautologous or allogenic tumor cells which express (endogenously orrecombaintly) a mutant p63, p53, or p73 polypeptide (or fragmentthereof), or lysates of such cells.

[0370] In clinical settings, the therapeutic compound of the presentinvention can be introduced into a patient by any of a number ofmethods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system or peptide can beintroduced systemically, e.g. by intravenous injection, and specifictransduction of the protein in the target cells occurs predominantlyfrom specificity of transfection provided by the gene delivery vehicle,cell-type or tissue-type expression due to the transcriptionalregulatory sequences controlling expression of the receptor gene, or acombination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle or peptide canbe introduced by catheter (see U.S. Pat. No. 5,328,470) or bystereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3054-3057). Avaccine gene can be delivered in a gene therapy construct byelectroporation using techniques described, for example, by Dev et al.((1994) Cancer Treat Rev 20:105-115).

[0371] The pharmaceutical preparation of the vaccine therapy constructor peptide can consist essentially of the gene delivery system in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery system can be produced intact from recombinant cells, e.g.retroviral or adenoviral vectors, the pharmaceutical preparation cancomprise one or more cells which produce the gene delivery system.

[0372] Suitable pharmaceutical vehicles for administration to a patientare known to those skilled in the art. For parenteral administration,the p63, p53, or p73 immunogen will usually be dissolved or suspended insterile water or saline. For enteral administration, the immunogen willbe incorporated into an inert carrier in tablet, liquid, or capsularform. The preparation may also be emulsified or the active ingredientencapsulated in liposome vehicles. The composition or formulation to beadministered will, in any event, contain a quantity of the p63, p53, orp73 polypeptide adequate to achieve the desired immunized state in thesubject being treated. The immunogen preparations according to theinvention may also contain other peptides or other immunogens.

[0373] Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.For instance, the immunogen can be formulated as a pharmaceuticallyacceptable acid- or base-addition salt, formed by reaction withinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid,and organic acids such as formic acid, acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid,succinic acid, maleic acid, and fumaric acid, or by reaction with aninorganic base such as sodium hydroxide, ammonium hydroxide, potassiumhydroxide, and organic bases such as mono-, di-, trialkyl and arylamines and substituted ethanolamines.

[0374] The immunogen, which may be coupled to a carrier, is preferablyadministered after being mixed with immunization adjuvants. Conventionaladjuvants include, for example, complete or incomplete Freund'sadjuvant, aluminum hydroxide, QuiI A, EMA, DDA, TDM-Squalene, lecithin,alum, saponin, and such other adjuvants as are well known to those inthe art, and also mixtures thereof. For example, the p63, p53, or p73immunogen may be mixed with the N-butyl ester (murabutide) of themuramyl dipeptide (MDP;N-acetyl-glucosamine-3-yl-acetyl-L-alanyl-D-isoglutamine) diluted in asaline solution. The mixture may then be emulsified by means of an equalvolume of squalene in the presence of arlacel (excipients). It is alsopossible to use other adjuvants such as analogues of MDP, bacterialfractions such as streptococcal preparations (OK 432), Biostim (01K2) ormodified lipopolysaccharide preparations (LPS), peptidoglycans (N-Opaca)or proteoglycans (K-Pneumonia). In the case of these excipients,water-in-oil emulsions are preferable to oil-in-water emulsions.

[0375] The use of the instant invention is predicted to be of benefit inthe treatment of any type of tumor which harbors a mutant p63, p53, orp734 gene. For example, treatment of tumors of the lung, breast, brain,bone, skin, bladder, kidney, ovary, or lymphocytes is contemplated. In apreferred embodiment the tumor vaccine of the present invention is usedto treat melanoma.

[0376] In addition to enhancing the immune response against a tumor atits original site, the tumor cell vaccine of the current invention mayalso be used in a method for preventing or treating metastatic spread ofa tumor or preventing or treating recurrence of a tumor. Thus,administration of modified tumor cells or modification of tumor cells invivo as described herein can provide tumor immunity against cells of theoriginal, unmodified tumor as well as metastases of the original tumoror possible regrowth of the original tumor.

[0377] Subjects develop an anti-tumor specific T cell response which isspecific for mutant forms of p63, p53, or p73 proteins and is effectivein keeping the patients disease free. Thus, the subject developsanti-tumor specific immunity. It is also contemplated that the inventionmay be useful in inducing immunity to tumors in susceptible individualsbefore they arise, for example in the case of familial malignancies. Astrong hereditary component has been identified for certain types ofmalignancies, for example certain breast and colon cancers and insusceptability to melanoma. In families with a known susceptibility to aparticular malignancy and in which one individual presently has a tumorbearing a mutant p63, p53, or p73 protein, peptides presented by class Imolecules of these patients could be administered to susceptible,histocompatible family members to induce an anti-tumor response in therecipient against the type of tumor to which the family is susceptible.This anti-tumor response could provide protective immunity to subsequentdevelopment of a tumor in the immunized recipient.

[0378] 4.9.1. Effective Dose

[0379] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining The Ld₅₀ (The Dose Lethal To50% Of The Population) And The Ed₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic induces arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0380] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0381] 4.9.2 Formulation and Use

[0382] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients. Thus, thecompounds and their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

[0383] For such therapy, the compounds of the invention can beformulated for a variety of loads of administration, including systemicand topical or localized administration. Techniques and formulationsgenerally may be found in Remmington's Pharmaceutical Sciences, MeadePublishing Co., Easton, Pa. For systemic administration, injection ispreferred, including intramuscular, intravenous, intraperitoneal, andsubcutaneous. For injection, the compounds of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, thecompounds may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

[0384] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0385] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound. For buccaladministration the compositions may take the form of tablets or lozengesformulated in conventional manner. For administration by inhalation, thecompounds for use according to the present invention are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0386] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0387] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0388] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0389] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration bile salts andfusidic acid derivatives. in addition, detergents may be used tofacilitate permeation. Transmucosal administration may be through nasalsprays or using suppositories. For topical administration, the oligomersof the invention are formulated into ointments, salves, gels, or creamsas generally known in the art. A wash solution can be used locally totreat an injury or inflammation to accelerate healing.

[0390] In clinical settings, a gene delivery system for the therapeuticp63 gene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance, a pharmaceuticalpreparation of the gene delivery system can be introduced systemically,e.g., by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al.(1994) PNAS 91: 3054-3057). A p63 gene, such as any one of the sequencesrepresented in the group consisting of SEQ ID NOS 1 and 3 or a sequencehomologous thereto can be delivered in a gene therapy construct byelectroporation using techniques described, for example, by Dev et al.((1994) Cancer Treat Rev 20:105-115).

[0391] The pharmaceutical preparation of the gene therapy construct orcompound of the invention can consist essentially of the gene deliverysystem in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle or compound is imbedded.Alternatively, where the complete gene delivery system can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

[0392] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0393] 4.9.3. Kits

[0394] The invention further provides kits for use in diagnostics orprognostic methods or for treating a disease or condition associatedwith an aberrant p63 protein. In one embodiment, the kit comprises apharmaceutical composition containing an effective amount of an p63antagonist therapeutic and instructions for use in treating orpreventing tumorigenesis. In yet another embodiment, the kit comprises apharmaceutical composition comprising an effective amount of an p63agonist therapeutic.

[0395] Yet other kits can be used to determine whether a subject has oris likely to develop a disease or condition associated with an aberrantp63 activity. Such a kit can comprise, e.g., one or more nucleic acidprobes capable of hybridizing specifically to at least a portion of anp63 gene or allelic variant thereof, or mutated form thereof.

[0396] 4.10. Additional Uses for p63 Proteins and Nucleic Acids

[0397] The p63 nucleic acids of the invention can further be used in thefollowing assays. The p63 gene can also be used as a chromosomal markerin genetic linkage studies involving genes other than p63.

[0398] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication are hereby expressly incorporated by reference. The practiceof the present invention will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I andII (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization(B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).All references cited herein are inorporated by reference in theirentirety.

EXEMPLIFICATION

[0399] The invention now being generally described, it will be morereadily understood by reference to the following examples which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention, and are not intended to limit theinvention.

EXAMPLES

[0400] I. Identification of Human and Murine p63

[0401] It has been obsreved that the intron-exon organization wasconserved between p73 and p53 (Kaghad et al., 1997), and from known exonand intron sizes for these two genes, it was possible to identify newmembers of this gene family using a PCR-based strategy of amplifying twoexons in a conserved domain and their intervening intron. Sequencesimilarity in exonic regions would demonstrate a related gene, whiledifferences in size and/or sequence or introns from p53 and p73 wouldindicate a novel family member. Non-degenerate and degenerate primerswere designed based in sequence homology among p53 and p73 cDNAs fromvarious species. Primers (5′-GGCCTCGAGTACAAITWCATGTGTAAYAG and5′GGCATCGATTCTCTTCCAGGGCAAGCACA), designed to anneal to regions in exon7 and exon 8, respectively, of p73 and p53, were used to amplifyproducts from human and mouse total genomic DNA with the followingconditions:

[0402] pre-PCR: 80° C. 2 min, add TAQ polymerase, 94° C. 5 min.

[0403] ‘Touchdown PCR’: 94° C. 1 min, 65° C. 1 min,, 72° C. 2 min for 3cycles: 94° 1 min, 64° C. 1 min, 72° C. 2 min for 3 cycles; 94° C. 1min, 63° C. 1 min, 72° C. 2 min for 3 cycles; 94° C. 1 min, 62° C. 1min, 72° C. 2 min for 2 cycles; 94° C. 1 min, 61° C. 1 min, 72° C. 2 minfor 2 cycles; 94° C. 1 min, 60° C. 1 min, 72° C. 2 min for 2 cycles; 94°C. 1 min, 59° C. 1 min, 72° C. 2 min for 2 cycles; 94° C. 1 min, 58° C.1 min, 72° C. 2 min for 20 cycles; 72° C. 7 min

[0404] The above generated amplicons of approximately 800 bp and 900 bpfrom human and mouse genomic DNA, respectively. Comparison with knownsizes of the corresponding intron (7) in p73 and p53 indicated thatthese amplicons were derived from a novel gene that shared homology inexonic regions, as demonstrated by their ability to be generated withthe above primers. The amplicons were then subcloned into a pCDNA3 orpCDNA3 or pCDNA3-GFP vector and sequenced using T7, SP6, and GFPprimers. Sequence analysis confirmed homology with regions in exon 7 andexon 8 of the p73 and p53 genes, showing strong nucleotide similarityand near identity at the amino acid level, particularly to p73.

[0405] II. Cloning of p63 via Exon-Bridging

[0406] The recent discovery of the p53-related gene, p73, suggested thatother members of the p53 gene family exist in mammalian genomes. Giventhe absence of p53-related sequences in available expressed sequence tag(EST) libraries, it was possible that p53-related genes were expressedat relatively low levels or in a tissue-restricted manner, and thatstandard hybridization or polymerase chain reaction (PCR) approacheswould be difficult. However, examination of the central portions of p53and p73 showed a remarkable conservation of intron positions, while thesize and sequences of these introns remained distinct between thesegenes (Kaghad et al., 1997; Yang et al., submitted). We thereforedesigned PCR primers that would anneal to regions in contiguous exonsconserved between p53 and p73 such that the amplification product wouldinclude the intervening intron. As products from the p53 and p73 geneswere predictable on the basis of known intron sizes and sequence, weanalyzed novel products for ones possessing terminal sequence homolocywith p53 and p73 bordering non-conserved intronic sequences. One primerpair designed to span intron 7 yielded an 800 basepair (bp) and 900 bpPCR product from human and mouse, respectively, genomic DNA (not shown).The ends of these products displayed sequence homology with the 3′ endof exon 7 and the 5′-end of exon 8 of p53 and p73, while the interveningsequence showed no homology with introns of either gene. The 800 bpamplicon derived from human genomic DNA was used as a hybridizationprobe to screen a human PAC library, which yielded a single clone of 120Kb. Similarly, the 900 bp amplicon from the mouse genomic DNA was usedto isolate a single, 16.5 Kb clone from a lambda DASH II murine genomicDNA phage library. Partial sequencing of exon 7 of the human and mousegenomic clones confirmed the presence of exonic sequences withsimilarity to those of p53 and p73, and yet the presence of third basecodon differences and of conserved substitutions, demonstrating thatthis gene was a novel member of the p53 family.

[0407] To deduce the amino acid sequence of the protein, or proteins,encoded by this gene, we constructed a cDNA library from E15 murineembryos lacking p73 and p53 genes (p73-/-,p53-/-; Yang et al.,unpublished results) and screened pools of the fractionated library byPCR and subsequent hybridization. Three full-length cDNAs were obtained,all of which shared a central domain with approximately 60 and 85% aminoacid identity with the DNA binding domains of p53 and p73, respectively(FIG. 1). Human cDNAs were deduced from sequencing reversetranscriptase-polymerase chain reaction (RT-PCR) products from theSK-N-MC neuroepithelioma cell line, and these revealed a remarkably highlevel of primary amino acid sequence conservation, bordering on 99%,with the mouse cDNAs (FIG. 1). Of the three murine cDNAs initiallyisolated, one encoded an acidic N-terminus similar to that required fortranscriptional activation by p53 and p73. Interestingly, the murinecDNA clone with the acidic N-terminus contains an additional 39 aminoacids upstream of the methionine start site seen in human sequences todate (FIGS. 1, 2). These N-termini have been denoted TA* for the longerN-terminus and TA for the sequence starting with the amino acid sequenceMSQ (FIGS. 1, 2). The other two murine cDNAs clones encoded proteinswith a truncated N-terminus (ΔN) lacking the acidic, putativetransactivation domain. Further transcript and cDNA analysis from bothmurine and human sources revealed the expression of additional variants.In total, at least six different transcripts, derived from alternativesplicing events and encoding proteins with two different N-termini(TA*/TA and ΔN) and three different C-termini (α, β, and γ), aredescribed (FIG. 2). Partial analysis of the murine and human genesindicated that the transcripts that give rise to the truncatedN-terminal proteins were derived from an alternative promoter andinitiation codon in intron 3 (exon 3′; FIG. 2A). Additionally, asplicing variant in exon 9 of both species alternatively deleting fouramino acids was detected in both species (not shown). To reflect thehigh degree of homology of the human and murine sequences to p53 andp73, as well as the immense complexity of these gene products withpredicted molecular weights ranging from 44,000 to 72,000 daltons, wepropose that this gene be called p63.

[0408] To map the human p63 gene, we employed fluorescence in situhybridization (FISH) techniques on human metaphase chromosome spreadsusing the human p63 PAC clone as a probe. The p63 PAC clone hybridizedto the long arm of chromosome 3, at approximately 3q27-29 (FIG. 3A). Wealso mapped the murine p63 gene using linkage analysis, which showedthat p63 is located on the proximal portion of chromosome 16 betweenanonymous DNA markers D 16Mit 1 and D16Mit3 (FIG. 3B). This region isknown to be syntenic with human chromosome 3q27-29, in agreement withthe in situ analysis of the human p63 gene.

[0409] Hup63geno (PAC) has since been deposited at the ATCC under theterms of the Budapest Treaty.

[0410] III. Mapping of Human p63 Gene

[0411] To map the human p63 gene, we used fluorescense in situhybridization (FISH) on human metaphase chromosome spreads using thehuman p63 PAC clone as a probe. The p63 PAC clone hybridized to the longarm of chromosome 3 at approximately 3q27-28 (FIG. 2A). Fluorescence insitu hybridization (FISH) was performed on human metaphase spreads usingHupo63geno (PAC) as a probe. The methods used protocols that are wellknown in the art. By this method, p63 was mapped to human chromosome3q27-29, a region implicated in various human syndromes including B-celllymphoma and large diffuse cell lymphoma.

[0412] IV. Cloning of Human p63 cDNAs

[0413] Sequence information for human p63 transcripts were obtained byRT-PCR on total RNA isolated from the SK-B-MC cell line.

[0414] V. Cloning of Murine p63 Gene

[0415] The 900 bp amplicon derived from mouse genomic DNA, describedabove, was used as a probe in hybridization screens for the murinehomolog of the p63 gene. Screening of a 129 mouse genomic phage library(in lambda DASH II) yielded a clone with a 16.5 kb insert containing themurine p63 gene. Hybridization screens were done as per standardprotocols, under the following conditions: prehybridization incubation(without probe) for 4 hrs at 50-55° C., in hybridization solution (50%formamide, 5×SSC, 2.5× Denhardts, 150 ug/ml salmon sperm DNA, 0.1% SDS);hybridization with ³²P-labeled probe (in hybridization solution) for 16hrs at 40° C.; washes done in 0.5×Ssc, 0.1% SDS at 50° C. The 16.5 kbinsert was released by a NotI digestion, subcloned into the pZerovector, and sequenced in its entirety. Sequence analysis showed theclone to contain a portion of the murine p63 gene, extending from intron4 through intron 10.

[0416] VI. Cloning of Murine p63 cDNAs

[0417] 5′ Rapid Amplification of cDNA Ends (RACE) was used to obtainfurther sequence information on p63 not contained within the murinegenomic clone. Total RNA was isolated from c15 embryos lacking both p53and p73, generated from mice bearing targeted mutations in both genes,and used as the template in a first stand cDNA synthesis reaction with amurine p63-specific primer (5′-GGCATCGATGAACTCACGGCTCAGCTC). An‘adapter’ primer (5′-TTTAGTGAGGGTTAATAAGCGGCCGCGTCGTGACTGGGAGCGC) wasthen ligated to the cDNA product using T4 RNA ligase. PCR wassubsequently performed on the ligation product using primers(5′-GCCCTGGAGGCGGCCGCTTATTAACCCTCAC and 5′-GGCATCGATGTAGACAGGCATGGCACG)with the conditions described in I. An approximately 610 bp amplicon wasgenerated, subcloned into pCDNA3 vector, and sequenced in its entirety.The 5′RACE product yielded a sequence corresponding to a N-terminaltruncated form of murine p63.

[0418] A bacterial plasmid cDNA library was constructed from mRNAisolated from e15 embryos lacking both p53 and p73, described above, andscreened for p63 cDNAs. Hybridization screens were done essentially asdescribed in V., using a probe, corresponding to exons 5 to 9 of p63,generated by RT-PCR on total p73-/-;p53-/- mouse RNA with primers(5′-GGGCTCGAGCTGAAGAAGCTGTACTGC and 5′-GGGATCGATCTCCGTTTCTTGATGGA).Three clones were identified and sequenced in their entirety. Thesecorresponded to three different, full-length splice variants of murinep63.

[0419] VII. Murine p63 Gene Targeting Vector

[0420] The 16.5 kb genomic fragment described in V. was used toconstruct a vector for targeted disruption of the p63 gene by homologousrecombination in murine embryonic stem (ES) cells. Briefly, the p63 genefragment was subcloned into the pBluescript SK(-) vector via a NotI/NarIdigestion followed by ligation to compatible cohesive ends generated bya NotI/ClaI digestion of the vector plasmid. This construct,pSK-murp63geno, was then digested with SpeI and ClaI to remove a 2900 bpregion corresponding to portions of intron 5, all of exon 6, intron 6,exon 7, intron 7, exon 8, and portions of intron 9 (note: exon andintron designations based on exon sequence homology with p73 and p53).This region was then replaced with the neomycin resistance gene undercontrol of the PGK promotor (PGK-NeoR), yielding pSK-murp63genoNeo. Thisplasmid was then digested with SacII/NheI, removing a 2200 bp fragmentcorresponding to a portion of intron 4 that was then replaced with theherpes simplex virus thymidine kinase gene (HSV-TK), yielding the finalp63 gene targeting vector, pSK-murp63ko. This vector will be linearizedand introduced into murine ES cells via electroporation. Doubleselection with G418 and gancyclovir, followed by DNA hybridization withexternal probes will identify ES clones which have under gone homologousrecombination. These will then be used in blastocyst reconstitutions togenerate chimeric mice bearing the targeted disruption in p63. Breedingof these chimeric mice with wildtype mice and intercrosses fromsubsequent F1 and F2 generations will yield mice deficient in one orboth copies of the p63 gene.

[0421] VIII. Cloning of Human p63 and Possible Novel, Related GenesUsing Murine p63 cDNA

[0422] We obtained a human genomic library enriched for chromosome 22from the ATCC and probed with a murine p63 cDNA fragment correspondingto exons 5 to 9, described in VI. Hybridizations were done as describedin V, but lower stringency washes were used (5×SSC/0.1% SDS). Thesescreens yielded one clone containing a 4 kb insert that was thensubcloned into pZero vector and sequenced in its entirety. Sequenceanalysis showed that this clone was identical to previously obtainedhuman p63 cDNAs in the corresponding exonic regions. This resultdemonstrated the ability to clone the human p63 gene using across-species (mouse) cDNA probe. Additional clones which yieldedpositive signals in our hybridization screens have been identified andwill be purified and sequenced to determine if they are novel members ofthe p73, p63, p53 family.

[0423] IX. Immunofluorescence

[0424] Transfection of baby hamster kidney (BHK) cells with myc-epitopetagged p63 cDNAs in pCDNAA3 vector and subsequent immunofluorescencedetection of protein was done essentially as described in Heald et al.,1993.

[0425] X. Expression of p63 in Human and Murine Tissues

[0426] As the p63 cDNAs were derived from murine embryos and human celllines, it was important to determine their expression in normal adulttissues. To address this issue, we immunized mice withbacterially-expressed glutathione-S-transferase-p63 fusion proteins, andproduced an array of monoclonal antibodies which recognize an epitopecommon to murine and human p63 and ΔN-p63 proteins (ExperimentalProcedures). Using one of these antibodies, the 4A4 clone, we probedparaffin sections of archival normal human tissues including foreskin,cervix, vaginal epithelium, urothelium, and prostate (FIGS. 4A-D). Inall of these tissues, the 4A4 monoclonal antibody showed strong nuclearstaining concentrated in the basal cells of the epithelium. Thepredominant localization of p63 to the basal layer of these stratifiedsquamous and transitional epithelia is interesting in that these cellsact as the progenitors of the suprabasal cells, which undergodifferentiation and cell death in regenerative epithelia (Jetten andHarvat, 1997). In the prostate, the relationship between thesep63-positive basal cells and the luminal cells is less well established,but it is thought that the basal cells are likewise the progenitors ofthe suprabasal, secretory cells (Myers and Grizzle, 1997).

[0427] To obtain a more extensive survey of p63 expression in adulttissues, we prepared total RNA from various murine tissues and performedRT-PCR reactions specific for the two different p63 N-termini, TA andΔN. This analysis revealed the presence of transcripts encoding TAp63variants in heart, testis, kidney/adrenal, thymus, brain (minuscerebellum), and cerebellum (FIG. 5a). The ΔNp63 transcript was detectedin the kidney/adrenal, spleen, and thymus, but absent from the heart,liver, testis, and brain, despite the normalization of template RNAconcentration in each reaction (FIGS. 5B,C). This RT-PCR analysisindicated that TAp63 and ΔNp63 transcripts are widely expressed in adulttissues.

[0428] The immunohistochemistry on human epithelial tissues revealedhigh levels of p63 expression in basal cells (FIG. 3). To determinewhich p63 isotypes were expressed in these cells, RT-PCR reactions wereperformed on RNA prepared from primary human foreskin keratinocytes,human ectocervix, and ME180 cervical carcinoma cells. While RNA derivedfrom the ME180 cells yielded a positive signal in the RT-PCR reactiondesigned to amplify transcripts encoding the acidic N-terminus, theprimary keratinocytes and the ectocervical cells showed little or noproduct (FIG. 5D, TA). In contrast, RNA from all three sources showedrobust signals in the RT-PCR reaction designed to amplify the ANp63transcript (FIG. 5D, ΔN).

[0429] The analysis of RNA from primary keratinocytes indicated that themajor p63 transcripts in these cells encoded ΔNp63 isotypes. To addressthis possibility at the protein level, we performed immunoblotting onprotein lysates derived from human primary foreskin keratinocytes (HFK)and ME180 cervical carcinoma cells with the 4A4 monoclonal antibody.Lysates of baby hamster kidney (BHK) cells transfected with mammalianexpression vectors encoding epitope-tagged p63 isotypes were included ascontrols for molecular weight comparison (FIG. 5E). Significantly, themajor product detected in primary keratinocytes and ME180 migrates atapproximately 8 okDa, slightly faster than the Myc epitope-taggedΔNp63α. (FIG. 5E). The ME180 cells also express a less abundant, thoughdetectable, product migrating at approximately 90-95 kDa, similar tothat of TAp63α (FIG. 5E). These data, taken together with the abundantΔNp63 RT-PCR product from the ME180 cell line and primary keratinocyteRNA, are consistent with notion that these epithelial cellspredominantly express the ΔNp63α isotype.

[0430] XI. Transactivation Functions of p63 Isotypes

[0431] The central domain of all p63 variants is highly homologous withthe DNA binding domains of p53 and p73, suggesting that at least somep63 isotypes function as transcriptional activators. The ΔNp63 variants,however, lack the N-terminal acidic residues thought to participate intransactivation functions of p53 and p73 (Ko and Prives, 1996; Levine,1997; Kaghad et al., 1997). To determine whether any of the p63 isotypescan act as transactivators, we tested their ability to induce expressionfrom a reporter gene under the control of a p53-responsive element. Sixp63 constructs, specifically TAp63α, TAp63γ, ΔNp63α, ΔNp63γ, TA*p63α,and TA*p63γ, as well as wildtype and mutant p53 expression vectors, wereseparately expressed in Saos-2 human osteosarcoma cells, which lackendogenous p53, along with a β-galactosidase reporter constructcontaining multiple copies of a minimal p53 binding sequence (PG-13, Kemet al., 1992). Lysates from cells expressing wildtype p53 yielded astrong β-galactosidase signal, while those expressing the p53 mutantshowed only a background signal (FIG. 6). Of the p63 isotypes tested inSaos-2 cells, only TAp63y exhibited strong transcriptional activation ofthe p53 reporter, with levels approaching 80% of that seen with p53.Interestingly, the TA*p63γ isotype, which has a 39 amino acid N-terminalextension not found in TAp63γ, proved to be a weak transactivator inSaos-2 cells, suggesting possible regulatory elements within thisadditional domain. As expected, neither the ΔNp63γ nor the ΔNp63αvariant, both of which lack the putative transctivation domain, showedstrong reporter activity (FIG. 6), although the ΔNp63γ gave a low butdetectable signal. Surprisingly, however, the ΔNp63α isotype also failedto yield a significant level of reporter gene expression, despite havingthe same transactivation domain as TAp63γ. A similar lack oftransactivation was seen with TA*p63α, pointing to additional regulatoryfacets of p63 involving its C-terminal domain.

[0432] XII. Induction of Apoptosis by p63 Isotypes

[0433] Expression of wildtype p53 induces apoptosis in a wide variety ofcells, whereas many p53 mutants have lost this ability (Oren, 1994). Todetermine whether any of the p63 isotypes possess similar death-inducingactivities, we compared the fates of baby hamster kidney (BHK) cellstransfected with p53 or p73 vectors with those expressing p63 isotypes.BHK cells were transfected with the p53, p73, and p63 expression vectorsand fixed 16 hours later. Cells were labeled using anti-Myc and anti-HAepitope tag antibodies to detect expressed proteins and with the DNAfluorochrome Hoechst 33278. Approximately 90% of the wildtypep53-expressing cells appeared raised from the substrate and showedhighly condensed, lobated nuclei characteristic of apoptotic cells (FIG.7A). In contrast, those expressing the p53(V143A) mutant showed a verylow percentage of apoptotic cells, despite high levels of exogenousprotein expression (FIG. 7B). Cells transfected with TAp637 provedhighly vulnerable to cell death, as evidenced by nuclear morphology,despite the generally low protein levels generated from exogenousexpression in these cells. However, an overexposure of the epitope-tagimmunofluorescence image showed a good correspondence between apoptoticcells and TAp63γ expression (FIG. 7C). Curiously, cells expressing theΔNp63γ isotype, which yielded low but measurable activity in theβ-galactosidase assays (FIG. 6), also exhibited a slight but noticeablelevel of apoptosis. The percentage of cell death induced by ΔNp63γhowever, was considerably less than those seen in cells expressing p53or TAp63γ, despite the fact that ΔNp63γ accumulates to very high levelsin BHK cells (FIG. 7D). Apoptosis was virtually absent in cellsexpressing high levels of either ΔNp63α (FIG. 7E), TAp63α, or TA*p63α,consistent with the lack of transactivation seen with these variants.Finally, p73α and p73β exhibited little or no apoptotic activity inthese cells at 16 hours, despite high levels of accumulation.

[0434] XIII. ΔN-p63γ Suppresses Transactivation by p53 and Enhances Thatof TA-p63γ

[0435] The ability of the TAp63 γ isotype to transactivate reportergenes bearing p53-responsive elements suggested that, in general, p63isotypes can interact with p53 DNA binding sites. As the ΔNp63γ isotypeslack the acidic N-terminus similar to that required for transcriptionalactivation by p53, it seemed feasible that such isotypes could act in adominant-negative manner towards both p53 and transactivating versionsof p63, such as TAp63γ. To test whether ΔNp63γ isotypes could in factsuppress the transactivation ability of p53, we transfected Saos-2 cellswith a constant amount wildtype p53 and varying concentrations of eitherΔNp63γ or ΔNp63α and assayed for transactivation of the PG-13 β-galreporter gene. At a 5:1 DNA ratio of p53 and ΔNp63γ transfected intoSaos-2 cells, reporter activity was reduced to 37% that of p53 alone,while a 1:1 ratio yielded less than 20% the transactivation of p53 (FIG.8A). ΔN-p63α also showed a similar, dose-dependent inhibition of p53transactivation, with the higher suppressor concentration (1:1) givingnear background (vector alone) levels of reporter signal (FIG. 8A).

[0436] We next asked if ΔN-p63 isotypes could likewise affecttransactivation by TAp63γ. Paradoxically, cells co-transfected withTAp63γ and ΔNp63γ (5:1) yielded reporter expression slightly above thatseen with TAp63γ alone (FIG. 8B). Higher levels of ΔNp63γ in thecotransfection (1:1 ratio) suppressed transactivation by TA-p63γ by amodest 20% (FIG. 8B). In contrast, ΔN-p63α proved to be a strongsuppressor of transactivation by TAp63γ, yielding only background levelsof reporter signal, even when co-transfected at one-fifth the DNAconcentration of TAp63γ (FIG. 8B).

[0437] Several mechanisms could underlie the ability of ΔNp63 isotypesto suppress p53 and p63 in these assays. For example, given the highdegree of sequence homology within the DNA binding domains of the p53and p63 proteins, it is likely that p63 can bind p53 DNA target sites ina competitive manner. To address this possibility, we asked whether p63isotypes, particularly those lacking detectable transactivationcapabilities, could nonetheless interact with p53 DNA binding sites.Electrophoretic mobility shift assays (EMSA) were performed using threeseparate oligonucelotides: a minimal p53 binding sequence site (PG), ap53 binding site in the p21 promoter (WAF), and a mutant p53 bindingsite (MG; Kern et al., 1992) with lysates of 293 human kidney cellstransfected with p53, ΔNp63γ, TAp63α and green fluorescent protein. p53,ΔNp63γ and TAp63α lysates all yielded significant shifts of both PG andWAF oligonucleotides, while GFP, included as a negative control, failedto display a similar shift (not shown). None of the lysates showed ashift of the control, non-p53 binding oligonucleotide, MG, thusdemonstrating the specificity of p53, ΔNp63γ and TAp63α. interactionswith the p53-binding sites.

[0438] Another mechanism by which p63 isotypes could affecttransactivation by p53 and p63 is via direct protein-proteininteractions, presumably through their oligomerization domains, Wetested the potential for such interactions using a glutathioneS-transferase (GST), TA*p63γ fusion construct (GST-TA*p63γ).Co-expression in BHK cells and subsequent binding assays showed strongassociations between GST-TA*p63γ and p63γ isotypes, including and ΔNDΔNp63α but failed to reveal an interaction with p53.

[0439] XIV. Electrophoretic Mobility Shift Assays (EMSA)

[0440] This invention provides nucleic acids encoding a DNA bindingdomain of a p63 cell regulator protein. Assays for determining thelocation of a DNA binding domain in proteins include gel retardationassays, well known in the art. Briefly, recombinant proteins comprisingvarious portions of a p63 cell regulator protein can be produced andtheir interaction with DNA can be measured by incubation with a DNAtarget sequence and separation of the complexes by gel electrophoresis.The DNA target sequence of a p63 cell regulator protein can bedetermined, e.g., by binding site selection experiments, well known inthe art. Binding site selection experiments are performed by incubationof a DNA binding protein, e.g., a p63 cell regulator protein with adegenerate pool of labeled double stranded oligonucleotides andisolation of the oligonucleotides which interact specifically with theDNA binding protein. Individual oligonucleotides are then sequenced.

[0441] EMSAs were performed essentially as described in Yang, A. et al.,(1998), Mol Cell 2, 305-316. Briefly, human 293 kidney cells weretransfected with p53, p63, and GFP expression vectors, as indicated inFIG. 25, using the calcium phosphate transfection method previouslydescribed (Heald et al., 1993, Cell 74, 463-474.; Yang et al., 1998).Cells were lysed in 150 μl detergent lysis buffer (50 mM Tris pH 8, 150mM NaCl, 0.1% Triton X-100) ˜24 hrs after transfection. Lysates werethen incubated for 1 hr at room temperature with 100 pM ³²Pradiolabeled, double-stranded oligonucleotides in binding buffer (16 mMHepes-KOH pH 7.5, 60 mM Kcl, 30 mM NaCl, 10% glycerol, 1 mMdithiothreitol, 10 μg/ml BSA). The following oligonucleotides were used,with annealing of oligonucleotide pairs performed prior to incubationwith lysate extracts above. PG: 5′-CCTGCCTGGACTTGCCTGG +5′-CCAGGCAAGTCCAGGCAGG. WAF: 5′-GAACATGTCCCAACATGTTG +5Õ-CAACATGTTGGGACATGTTC. MG: 5′-CCTTAATGGACTTTAATGG +5Õ-CCATTAAAGTCCATTAAGG.

[0442] XV. Induction of p63 in Response to UV/DNA Damage

[0443] These experiments show that like p53 p63 is induced in responseto stress signals such as UV-DNA damage.

[0444] RT-PCR Analysis

[0445] Total RNA was isolated from tissues and cell lines using RNAzol,dissolved in 10 mM Tris pH8, ImMEDTA (TE), and quantified usingultraviolet absorption at 260 nm. RT-PCR reactions were performed withthe One-Step RT-PCR kit (Gibco-BRL), using 0.25 ug total RNA in 25 ulreactions under the following conditions: 50° C. 30 min; 94° C. 2 min;94° C. 30 sec, 52° C. 30 sec, 72° C. 1 min for 40 cycles; 72° C. 5 min.The following primers were used: human p63 TA-specific reaction:5′-ATGTCCCAGAGCCACACAG and 5′-AGCTCATGGTTGGGGCAC; human p63 ▴N-specificreaction: 5′-CAGACTCAATTTAGTGAG and 5′-AGCTCATGGTTGGGGCAC

[0446] UV-Irradiation of Human Keratinocytes

[0447] Human foreskin primary keratinocytes were cultured inKeratinocyte-SFM media (Gibco-BRL) and maintained in 5% CO₂. Thekeratinocytes were treated with 300 J/m² UV irradiation, and harvestedat times indicated for total RNA using RNAzol, as described above. RNAfrom untreated keratinocytes obtained from the same culture was used asa control.

[0448] Differentiation of Human Keratinocytes

[0449] Human foreskin primary keratinocytes were cultured inKeratinocyte-SFM media (Gibco-BRL) and maintained in 5% CO₂. To inducedifferentiation, Keratinocyte-SFM media was replaced with DMEM media(Gibco-BRL) containing 10% fetal bovine serum (FBS). Cells wereharvested for RNA after addition of DMEM/10% FBS at times indicated. RNAfrom untreated keratinocytes obtained from the same culture was used asa control.

[0450] XVI. Screening for Mutations of the p63 Gene in Human Tumors andDiseases.

[0451] Direct sequencing, using standard techniques, of the p63 genewill yield information on the genomic organization (i.e. intron/exonboundaries) and nucleotide sequence for exons, introns and promoterregions of p63. An important biological and diagnostic application forthese data will be to screen for sequence mutations and/or polymorphisms(including nucleotide substitutions, insertions, or deletions) that mayresult in a loss or gain of function of the p63 gene. These screens willemploy the use of techniques standard in the field, including, but notrestricted to, single strand conformation polymorphism (SSCP) analysis,and direct sequencing of DNA or RNA samples obtained from patients.

[0452] As one means of enabling this application, we have isolated a PACclone of an approximately 120 kilobase genomic segment containing thep63 gene. Briefly, an 800 bp amplicon, derived from PCR on human genomicDNA and corresponding to portions of exon 7 and 8 and interveningintron, was used as a probe in hybridization screens for the human p63gene. Screening (done by Genome Systems) of a human genomic PAC library(made from white blood cells, male) yielded one clone containing the p63gene. We have confirmed the identity of this PAC clone using by PCR.Hup63geno (PAC) has been deposited at the ATCC under the terms of theBudapest Treaty. The Hup63geno (PAC) clone likely contains a majority ofthe p63 gene, as the DNA probe used hybridizes to a core, central domainof the gene. Regardless, the sequence information, as well as the use ofDNA probes derived from this PAC clone render the isolation of anyportion of the p63 gene missing from this clone a standard and obviousapplication.

[0453] XVII. Lineage-Specific Expression of p63 in Genital TractNeoplasia Vulvar, cervical, endometrial, and ovarian epithelialneoplasms, mixed mullerian tumors (MMMT), stromal sarcomas and adjacentnormal epithelia were studied. Serial sections were stained withmonoclonal antibodies to p63 and p53. Percentages of cells staining wereestimated for each neoplastic phenotype. It was found that in the vulvaor cervix, p63 expression was limited to squamous epithelium and reservecell populations. Staining was uniformly negative in benign andneoplastic endocervical glandular epithelium. Staining was weak (lessthan 10%) or absent in all but 2 endometrial adenocarcinomas, in allMMMTs, and in all ovarian neoplasms except one low grade transitionalcarcinoma. When present in adenocarcinomas, p63 staining predominated inbasal/reserve type cells and foci of squamous metaplasia. P53 expressionwas conspicuous (≧10%) only in endometrial serous carcinomas (12/16),one stromal sarcoma, and one MMMT, and did not co-localize with p63.

[0454] Therefore, it appears that p63 is a unique homologue of p53which, in the cervix, is expressed exclusively in squamous epithelium orreserve cells. In glandular lesions of other sites, p63 predominates inreserve cells, areas of squamous differentiation and basal cellpopulations. The sharp differences in expression of p63 betweenglandular and squamous epithelium, particularly in the cervix, mayprovide insights into the mechanisms determining phenotype in bothbenign and neoplastic epithelial proliferations.

[0455] XVIII. P63 is a Differentiation Specific Marker in CervicalSquamous Epithelium

[0456] The distribution of p63 expression in a range of cervicalsquamous epithelia was examined and contrasted it with markers for cellproliferation (Ki-67) because p63 is homologous to p53 and p53regulation has been implicated in the pathogenesis of HPV-relatedsquamous neoplasia.

[0457] 31 biopsies classified as reactive, atrophic, intraepithelial andinvasive cervical squamous epithelial alterations, as well as normalmucosa, were analyzed by immunohistochemistry for p53 and Ki-67 fordistribution and correlation with morphologic phenotype. It was foundthat distribution of p63 closely paralleled squamous celldifferentiation, staining all nuclei in the lower one third to one halfof normal squamous epithelium only. Diffuse staining of all epithelialcells occurred in immature epithelia, including atrophy, immaturemetaplasia, immature LSILs (papillary immature metaplasia) and theimmature cells of conventional low and high grade SIL and invasivecancer. In contrast, Ki-67 staining was more diffuse in neoplasticlesions, being expressed in both differentiated and undifferentiatedcell nuclei, and less frequently expressed in benign processes.

[0458] Therefore, it appears that p63 is a unique homologue of p53which, in the cervix, is expressed almost exclusively in immaturesquamous epithelium irrespective of the pathologic process. Themorphological and immunohistochemical evidence are consistent with arole of p63 in cell differentiation, uncoupled from both cellproliferation and HPV expression. Cessation or down-regulation of p63expression may play a critical role in the process of squamousdifferentiation, both benign and neoplastic.

1 50 1 1926 DNA Homo sapiens CDS (1)..(1923) 1 atg tcc cag agc aca cagaca aat gaa ttc ctc agt cca gag gtt ttc 48 Met Ser Gln Ser Thr Gln ThrAsn Glu Phe Leu Ser Pro Glu Val Phe 1 5 10 15 cag cat atc tgg gat tttctg gaa cag cct ata tgt tca gtt cag ccc 96 Gln His Ile Trp Asp Phe LeuGlu Gln Pro Ile Cys Ser Val Gln Pro 20 25 30 att gac ttg aac ttt gtg gatgaa cca tca gaa gat ggt gcg aca aac 144 Ile Asp Leu Asn Phe Val Asp GluPro Ser Glu Asp Gly Ala Thr Asn 35 40 45 aag att gag att agc atg gac tgtatc cgc atg cag gac tcg gac ctg 192 Lys Ile Glu Ile Ser Met Asp Cys IleArg Met Gln Asp Ser Asp Leu 50 55 60 agt gac ccc atg tgg cca cag tac acgaac ctg ggg ctc ctg aac agc 240 Ser Asp Pro Met Trp Pro Gln Tyr Thr AsnLeu Gly Leu Leu Asn Ser 65 70 75 80 atg gac cag cag att cag aac ggc tcctcg tcc acc agt ccc tat aac 288 Met Asp Gln Gln Ile Gln Asn Gly Ser SerSer Thr Ser Pro Tyr Asn 85 90 95 aca gac cac gcg cag aac agc gtc acg gcgccc tcg ccc tac gca cag 336 Thr Asp His Ala Gln Asn Ser Val Thr Ala ProSer Pro Tyr Ala Gln 100 105 110 ccc agc tcc acc ttc gat gct ctc tct ccatca ccc gcc atc ccc tcc 384 Pro Ser Ser Thr Phe Asp Ala Leu Ser Pro SerPro Ala Ile Pro Ser 115 120 125 aac acc gac tac cca ggc ccg cac agt ttcgac gtg tcc ttc cag cag 432 Asn Thr Asp Tyr Pro Gly Pro His Ser Phe AspVal Ser Phe Gln Gln 130 135 140 tcg agc acc gcc aag tcg gcc acc tgg acgtat tcc act gaa ctg aag 480 Ser Ser Thr Ala Lys Ser Ala Thr Trp Thr TyrSer Thr Glu Leu Lys 145 150 155 160 aaa ctc tac tgc caa att gca aag acatgc ccc atc cag atc aag gtg 528 Lys Leu Tyr Cys Gln Ile Ala Lys Thr CysPro Ile Gln Ile Lys Val 165 170 175 atg acc cca cct cct cag gga gct gttatc cgc gcc atg cct gtc tac 576 Met Thr Pro Pro Pro Gln Gly Ala Val IleArg Ala Met Pro Val Tyr 180 185 190 aaa aaa gct gag cac gtc acg gag gtggtg aag cgg tgc ccc aac cat 624 Lys Lys Ala Glu His Val Thr Glu Val ValLys Arg Cys Pro Asn His 195 200 205 gag ctg agc cgt gaa ttc aac gag ggacag att gcc cct cct agt cat 672 Glu Leu Ser Arg Glu Phe Asn Glu Gly GlnIle Ala Pro Pro Ser His 210 215 220 ttg att cga gta gag ggg aac agc catgcc cag tat gta gaa gat ccc 720 Leu Ile Arg Val Glu Gly Asn Ser His AlaGln Tyr Val Glu Asp Pro 225 230 235 240 atc aca gga aga cag agt gtg ctggta cct tat gag cca ccc cag gtt 768 Ile Thr Gly Arg Gln Ser Val Leu ValPro Tyr Glu Pro Pro Gln Val 245 250 255 ggc act gaa ttc acg aca gtc ttgtac aat ttc atg tgt aac agc agt 816 Gly Thr Glu Phe Thr Thr Val Leu TyrAsn Phe Met Cys Asn Ser Ser 260 265 270 tgt gtt gga ggg atg aac cgc cgtcca att tta atc att gtt act ctg 864 Cys Val Gly Gly Met Asn Arg Arg ProIle Leu Ile Ile Val Thr Leu 275 280 285 gaa acc aga gat ggg caa gtc ctgggc cga cgc tgc ttt gag gcc cgg 912 Glu Thr Arg Asp Gly Gln Val Leu GlyArg Arg Cys Phe Glu Ala Arg 290 295 300 atc tgt gct tgc cca gga aga gacagg aag gcg gat gaa gat agc atc 960 Ile Cys Ala Cys Pro Gly Arg Asp ArgLys Ala Asp Glu Asp Ser Ile 305 310 315 320 aga aag cag caa gtt tcg gacagt aca aag aac ggt gat ggt acg aag 1008 Arg Lys Gln Gln Val Ser Asp SerThr Lys Asn Gly Asp Gly Thr Lys 325 330 335 cgc ccg ttt cgt cag aac acacat ggt atc cag atg aca tcc atc aag 1056 Arg Pro Phe Arg Gln Asn Thr HisGly Ile Gln Met Thr Ser Ile Lys 340 345 350 aaa cga aga tcc cca gat gatgaa ctg tta tac tta cca gtg agg ggc 1104 Lys Arg Arg Ser Pro Asp Asp GluLeu Leu Tyr Leu Pro Val Arg Gly 355 360 365 cgt gag act tat gaa atg ctgttg aag atc aaa gag tcc ctg gaa ctc 1152 Arg Glu Thr Tyr Glu Met Leu LeuLys Ile Lys Glu Ser Leu Glu Leu 370 375 380 atg cag tac ctt cct cag cacaca att gaa acg tac agg caa cag caa 1200 Met Gln Tyr Leu Pro Gln His ThrIle Glu Thr Tyr Arg Gln Gln Gln 385 390 395 400 cag cag cag cac cag cactta ctt cag aaa cag acc tca ata cag tct 1248 Gln Gln Gln His Gln His LeuLeu Gln Lys Gln Thr Ser Ile Gln Ser 405 410 415 cca tct tca tat ggt aacagc tcc cca cct ctg aac aaa atg aac agc 1296 Pro Ser Ser Tyr Gly Asn SerSer Pro Pro Leu Asn Lys Met Asn Ser 420 425 430 atg aac aag ctg cct tctgtg agc cag ctt atc aac cct cag cag cgc 1344 Met Asn Lys Leu Pro Ser ValSer Gln Leu Ile Asn Pro Gln Gln Arg 435 440 445 aac gcc ctc act cct acaacc att cct gat ggc atg gga gcc aac att 1392 Asn Ala Leu Thr Pro Thr ThrIle Pro Asp Gly Met Gly Ala Asn Ile 450 455 460 ccc atg atg ggc acc cacatg cca atg gct gga gac atg aat gga ctc 1440 Pro Met Met Gly Thr His MetPro Met Ala Gly Asp Met Asn Gly Leu 465 470 475 480 agc ccc acc cag gcactc cct ccc cca ctc tcc atg cca tcc acc tcc 1488 Ser Pro Thr Gln Ala LeuPro Pro Pro Leu Ser Met Pro Ser Thr Ser 485 490 495 cac tgc aca ccc ccacct ccg tat ccc aca gat tgc agc att gtc agt 1536 His Cys Thr Pro Pro ProPro Tyr Pro Thr Asp Cys Ser Ile Val Ser 500 505 510 ttc tta gcg agg ttgggc tgt tca tca tgt ctg gac tat ttc acg acc 1584 Phe Leu Ala Arg Leu GlyCys Ser Ser Cys Leu Asp Tyr Phe Thr Thr 515 520 525 cag ggg ctg acc accatc tat cag att gag cat tac tcc atg gat gat 1632 Gln Gly Leu Thr Thr IleTyr Gln Ile Glu His Tyr Ser Met Asp Asp 530 535 540 ctg gca agt ctg aaaatc cct gag caa ttt cga cat gcg atc tgg aag 1680 Leu Ala Ser Leu Lys IlePro Glu Gln Phe Arg His Ala Ile Trp Lys 545 550 555 560 ggc atc ctg gaccac cgg cag ctc cac gaa ttc tcc tcc cct tct cat 1728 Gly Ile Leu Asp HisArg Gln Leu His Glu Phe Ser Ser Pro Ser His 565 570 575 ctc ctg cgg acccca agc agt gcc tct aca gtc agt gtg ggc tcc agt 1776 Leu Leu Arg Thr ProSer Ser Ala Ser Thr Val Ser Val Gly Ser Ser 580 585 590 gag acc cgg ggtgag cgt gtt att gat gct gtg cga ttc acc ctc cgc 1824 Glu Thr Arg Gly GluArg Val Ile Asp Ala Val Arg Phe Thr Leu Arg 595 600 605 cag acc atc tctttc cca ccc cga gat gag tgg aat gac ttc aac ttt 1872 Gln Thr Ile Ser PhePro Pro Arg Asp Glu Trp Asn Asp Phe Asn Phe 610 615 620 gac atg gat gctcgc cgc aat aag caa cag cgc atc aaa gag gag ggg 1920 Asp Met Asp Ala ArgArg Asn Lys Gln Gln Arg Ile Lys Glu Glu Gly 625 630 635 640 gag tga 1926Glu 2 1551 DNA Homo sapiens CDS (1)..(1548) 2 atg tcc cag agc aca cagaca aat gaa ttc ctc agt cca gag gtt ttc 48 Met Ser Gln Ser Thr Gln ThrAsn Glu Phe Leu Ser Pro Glu Val Phe 1 5 10 15 cag cat atc tgg gat tttctg gaa cag cct ata tgt tca gtt cag ccc 96 Gln His Ile Trp Asp Phe LeuGlu Gln Pro Ile Cys Ser Val Gln Pro 20 25 30 att gac ttg aac ttt gtg gatgaa cca tca gaa gat ggt gcg aca aac 144 Ile Asp Leu Asn Phe Val Asp GluPro Ser Glu Asp Gly Ala Thr Asn 35 40 45 aag att gag att agc atg gac tgtatc cgc atg cag gac tcg gac ctg 192 Lys Ile Glu Ile Ser Met Asp Cys IleArg Met Gln Asp Ser Asp Leu 50 55 60 agt gac ccc atg tgg cca cag tac acgaac ctg ggg ctc ctg aac agc 240 Ser Asp Pro Met Trp Pro Gln Tyr Thr AsnLeu Gly Leu Leu Asn Ser 65 70 75 80 atg gac cag cag att cag aac ggc tcctcg tcc acc agt ccc tat aac 288 Met Asp Gln Gln Ile Gln Asn Gly Ser SerSer Thr Ser Pro Tyr Asn 85 90 95 aca gac cac gcg cag aac agc gtc acg gcgccc tcg ccc tac gca cag 336 Thr Asp His Ala Gln Asn Ser Val Thr Ala ProSer Pro Tyr Ala Gln 100 105 110 ccc agc tcc acc ttc gat gct ctc tct ccatca ccc gcc atc ccc tcc 384 Pro Ser Ser Thr Phe Asp Ala Leu Ser Pro SerPro Ala Ile Pro Ser 115 120 125 aac acc gac tac cca ggc ccg cac agt ttcgac gtg tcc ttc cag cag 432 Asn Thr Asp Tyr Pro Gly Pro His Ser Phe AspVal Ser Phe Gln Gln 130 135 140 tcg agc acc gcc aag tcg gcc acc tgg acgtat tcc act gaa ctg aag 480 Ser Ser Thr Ala Lys Ser Ala Thr Trp Thr TyrSer Thr Glu Leu Lys 145 150 155 160 aaa ctc tac tgc caa att gca aag acatgc ccc atc cag atc aag gtg 528 Lys Leu Tyr Cys Gln Ile Ala Lys Thr CysPro Ile Gln Ile Lys Val 165 170 175 atg acc cca cct cct cag gga gct gttatc cgc gcc atg cct gtc tac 576 Met Thr Pro Pro Pro Gln Gly Ala Val IleArg Ala Met Pro Val Tyr 180 185 190 aaa aaa gct gag cac gtc acg gag gtggtg aag cgg tgc ccc aac cat 624 Lys Lys Ala Glu His Val Thr Glu Val ValLys Arg Cys Pro Asn His 195 200 205 gag ctg agc cgt gaa ttc aac gag ggacag att gcc cct cct agt cat 672 Glu Leu Ser Arg Glu Phe Asn Glu Gly GlnIle Ala Pro Pro Ser His 210 215 220 ttg att cga gta gag ggg aac agc catgcc cag tat gta gaa gat ccc 720 Leu Ile Arg Val Glu Gly Asn Ser His AlaGln Tyr Val Glu Asp Pro 225 230 235 240 atc aca gga aga cag agt gtg ctggta cct tat gag cca ccc cag gtt 768 Ile Thr Gly Arg Gln Ser Val Leu ValPro Tyr Glu Pro Pro Gln Val 245 250 255 ggc act gaa ttc acg aca gtc ttgtac aat ttc atg tgt aac agc agt 816 Gly Thr Glu Phe Thr Thr Val Leu TyrAsn Phe Met Cys Asn Ser Ser 260 265 270 tgt gtt gga ggg atg aac cgc cgtcca att tta atc att gtt act ctg 864 Cys Val Gly Gly Met Asn Arg Arg ProIle Leu Ile Ile Val Thr Leu 275 280 285 gaa acc aga gat ggg caa gtc ctgggc cga cgc tgc ttt gag gcc cgg 912 Glu Thr Arg Asp Gly Gln Val Leu GlyArg Arg Cys Phe Glu Ala Arg 290 295 300 atc tgt gct tgc cca gga aga gacagg aag gcg gat gaa gat agc atc 960 Ile Cys Ala Cys Pro Gly Arg Asp ArgLys Ala Asp Glu Asp Ser Ile 305 310 315 320 aga aag cag caa gtt tcg gacagt aca aag aac ggt gat ggt acg aag 1008 Arg Lys Gln Gln Val Ser Asp SerThr Lys Asn Gly Asp Gly Thr Lys 325 330 335 cgc ccg ttt cgt cag aac acacat ggt atc cag atg aca tcc atc aag 1056 Arg Pro Phe Arg Gln Asn Thr HisGly Ile Gln Met Thr Ser Ile Lys 340 345 350 aaa cga aga tcc cca gat gatgaa ctg tta tac tta cca gtg agg ggc 1104 Lys Arg Arg Ser Pro Asp Asp GluLeu Leu Tyr Leu Pro Val Arg Gly 355 360 365 cgt gag act tat gaa atg ctgttg aag atc aaa gag tcc ctg gaa ctc 1152 Arg Glu Thr Tyr Glu Met Leu LeuLys Ile Lys Glu Ser Leu Glu Leu 370 375 380 atg cag tac ctt cct cag cacaca att gaa acg tac agg caa cag caa 1200 Met Gln Tyr Leu Pro Gln His ThrIle Glu Thr Tyr Arg Gln Gln Gln 385 390 395 400 cag cag cag cac cag cactta ctt cag aaa cag acc tca ata cag tct 1248 Gln Gln Gln His Gln His LeuLeu Gln Lys Gln Thr Ser Ile Gln Ser 405 410 415 cca tct tca tat ggt aacagc tcc cca cct ctg aac aaa atg aac agc 1296 Pro Ser Ser Tyr Gly Asn SerSer Pro Pro Leu Asn Lys Met Asn Ser 420 425 430 atg aac aag ctg cct tctgtg agc cag ctt atc aac cct cag cag cgc 1344 Met Asn Lys Leu Pro Ser ValSer Gln Leu Ile Asn Pro Gln Gln Arg 435 440 445 aac gcc ctc act cct acaacc att cct gat ggc atg gga gcc aac att 1392 Asn Ala Leu Thr Pro Thr ThrIle Pro Asp Gly Met Gly Ala Asn Ile 450 455 460 ccc atg atg ggc acc cacatg cca atg gct gga gac atg aat gga ctc 1440 Pro Met Met Gly Thr His MetPro Met Ala Gly Asp Met Asn Gly Leu 465 470 475 480 agc ccc acc cag gcactc cct ccc cca ctc tcc atg cca tcc acc tcc 1488 Ser Pro Thr Gln Ala LeuPro Pro Pro Leu Ser Met Pro Ser Thr Ser 485 490 495 cac tgc aca ccc ccacct ccg tat ccc aca gat tgc agc att gtc agg 1536 His Cys Thr Pro Pro ProPro Tyr Pro Thr Asp Cys Ser Ile Val Arg 500 505 510 atc tgg caa gtc tga1551 Ile Trp Gln Val 515 3 1347 DNA Homo sapiens CDS (1)..(1344) 3 atgtcc cag agc aca cag aca aat gaa ttc ctc agt cca gag gtt ttc 48 Met SerGln Ser Thr Gln Thr Asn Glu Phe Leu Ser Pro Glu Val Phe 1 5 10 15 cagcat atc tgg gat ttt ctg gaa cag cct ata tgt tca gtt cag ccc 96 Gln HisIle Trp Asp Phe Leu Glu Gln Pro Ile Cys Ser Val Gln Pro 20 25 30 att gacttg aac ttt gtg gat gaa cca tca gaa gat ggt gcg aca aac 144 Ile Asp LeuAsn Phe Val Asp Glu Pro Ser Glu Asp Gly Ala Thr Asn 35 40 45 aag att gagatt agc atg gac tgt atc cgc atg cag gac tcg gac ctg 192 Lys Ile Glu IleSer Met Asp Cys Ile Arg Met Gln Asp Ser Asp Leu 50 55 60 agt gac ccc atgtgg cca cag tac acg aac ctg ggg ctc ctg aac agc 240 Ser Asp Pro Met TrpPro Gln Tyr Thr Asn Leu Gly Leu Leu Asn Ser 65 70 75 80 atg gac cag cagatt cag aac ggc tcc tcg tcc acc agt ccc tat aac 288 Met Asp Gln Gln IleGln Asn Gly Ser Ser Ser Thr Ser Pro Tyr Asn 85 90 95 aca gac cac gcg cagaac agc gtc acg gcg ccc tcg ccc tac gca cag 336 Thr Asp His Ala Gln AsnSer Val Thr Ala Pro Ser Pro Tyr Ala Gln 100 105 110 ccc agc tcc acc ttcgat gct ctc tct cca tca ccc gcc atc ccc tcc 384 Pro Ser Ser Thr Phe AspAla Leu Ser Pro Ser Pro Ala Ile Pro Ser 115 120 125 aac acc gac tac ccaggc ccg cac agt ttc gac gtg tcc ttc cag cag 432 Asn Thr Asp Tyr Pro GlyPro His Ser Phe Asp Val Ser Phe Gln Gln 130 135 140 tcg agc acc gcc aagtcg gcc acc tgg acg tat tcc act gaa ctg aag 480 Ser Ser Thr Ala Lys SerAla Thr Trp Thr Tyr Ser Thr Glu Leu Lys 145 150 155 160 aaa ctc tac tgccaa att gca aag aca tgc ccc atc cag atc aag gtg 528 Lys Leu Tyr Cys GlnIle Ala Lys Thr Cys Pro Ile Gln Ile Lys Val 165 170 175 atg acc cca cctcct cag gga gct gtt atc cgc gcc atg cct gtc tac 576 Met Thr Pro Pro ProGln Gly Ala Val Ile Arg Ala Met Pro Val Tyr 180 185 190 aaa aaa gct gagcac gtc acg gag gtg gtg aag cgg tgc ccc aac cat 624 Lys Lys Ala Glu HisVal Thr Glu Val Val Lys Arg Cys Pro Asn His 195 200 205 gag ctg agc cgtgaa ttc aac gag gga cag att gcc cct cct agt cat 672 Glu Leu Ser Arg GluPhe Asn Glu Gly Gln Ile Ala Pro Pro Ser His 210 215 220 ttg att cga gtagag ggg aac agc cat gcc cag tat gta gaa gat ccc 720 Leu Ile Arg Val GluGly Asn Ser His Ala Gln Tyr Val Glu Asp Pro 225 230 235 240 atc aca ggaaga cag agt gtg ctg gta cct tat gag cca ccc cag gtt 768 Ile Thr Gly ArgGln Ser Val Leu Val Pro Tyr Glu Pro Pro Gln Val 245 250 255 ggc act gaattc acg aca gtc ttg tac aat ttc atg tgt aac agc agt 816 Gly Thr Glu PheThr Thr Val Leu Tyr Asn Phe Met Cys Asn Ser Ser 260 265 270 tgt gtt ggaggg atg aac cgc cgt cca att tta atc att gtt act ctg 864 Cys Val Gly GlyMet Asn Arg Arg Pro Ile Leu Ile Ile Val Thr Leu 275 280 285 gaa acc agagat ggg caa gtc ctg ggc cga cgc tgc ttt gag gcc cgg 912 Glu Thr Arg AspGly Gln Val Leu Gly Arg Arg Cys Phe Glu Ala Arg 290 295 300 atc tgt gcttgc cca gga aga gac agg aag gcg gat gaa gat agc atc 960 Ile Cys Ala CysPro Gly Arg Asp Arg Lys Ala Asp Glu Asp Ser Ile 305 310 315 320 aga aagcag caa gtt tcg gac agt aca aag aac ggt gat ggt acg aag 1008 Arg Lys GlnGln Val Ser Asp Ser Thr Lys Asn Gly Asp Gly Thr Lys 325 330 335 cgc ccgttt cgt cag aac aca cat ggt atc cag atg aca tcc atc aag 1056 Arg Pro PheArg Gln Asn Thr His Gly Ile Gln Met Thr Ser Ile Lys 340 345 350 aaa cgaaga tcc cca gat gat gaa ctg tta tac tta cca gtg agg ggc 1104 Lys Arg ArgSer Pro Asp Asp Glu Leu Leu Tyr Leu Pro Val Arg Gly 355 360 365 cgt gagact tat gaa atg ctg ttg aag atc aaa gag tcc ctg gaa ctc 1152 Arg Glu ThrTyr Glu Met Leu Leu Lys Ile Lys Glu Ser Leu Glu Leu 370 375 380 atg cagtac ctt cct cag cac aca att gaa acg tac agg caa cag caa 1200 Met Gln TyrLeu Pro Gln His Thr Ile Glu Thr Tyr Arg Gln Gln Gln 385 390 395 400 cagcag cag cac cag cac tta ctt cag aaa cat ctc ctt tca gcc tgc 1248 Gln GlnGln His Gln His Leu Leu Gln Lys His Leu Leu Ser Ala Cys 405 410 415 ttcagg aat gag ctt gtg gag ccc cgg aga gaa act cca aaa caa tct 1296 Phe ArgAsn Glu Leu Val Glu Pro Arg Arg Glu Thr Pro Lys Gln Ser 420 425 430 gacgtc ttc ttt aga cat tcc aag ccc cca aac cga tca gtg tac cca 1344 Asp ValPhe Phe Arg His Ser Lys Pro Pro Asn Arg Ser Val Tyr Pro 435 440 445 tag1347 4 1761 DNA Homo sapiens CDS (1)..(1758) 4 atg ttg tac ctg gaa aacaat gcc cag act caa ttt agt gag cca cag 48 Met Leu Tyr Leu Glu Asn AsnAla Gln Thr Gln Phe Ser Glu Pro Gln 1 5 10 15 tac acg aac ctg ggg ctcctg aac agc atg gac cag cag att cag aac 96 Tyr Thr Asn Leu Gly Leu LeuAsn Ser Met Asp Gln Gln Ile Gln Asn 20 25 30 ggc tcc tcg tcc acc agt ccctat aac aca gac cac gcg cag aac agc 144 Gly Ser Ser Ser Thr Ser Pro TyrAsn Thr Asp His Ala Gln Asn Ser 35 40 45 gtc acg gcg ccc tcg ccc tac gcacag ccc agc tcc acc ttc gat gct 192 Val Thr Ala Pro Ser Pro Tyr Ala GlnPro Ser Ser Thr Phe Asp Ala 50 55 60 ctc tct cca tca ccc gcc atc ccc tccaac acc gac tac cca ggc ccg 240 Leu Ser Pro Ser Pro Ala Ile Pro Ser AsnThr Asp Tyr Pro Gly Pro 65 70 75 80 cac agt ttc gac gtg tcc ttc cag cagtcg agc acc gcc aag tcg gcc 288 His Ser Phe Asp Val Ser Phe Gln Gln SerSer Thr Ala Lys Ser Ala 85 90 95 acc tgg acg tat tcc act gaa ctg aag aaactc tac tgc caa att gca 336 Thr Trp Thr Tyr Ser Thr Glu Leu Lys Lys LeuTyr Cys Gln Ile Ala 100 105 110 aag aca tgc ccc atc cag atc aag gtg atgacc cca cct cct cag gga 384 Lys Thr Cys Pro Ile Gln Ile Lys Val Met ThrPro Pro Pro Gln Gly 115 120 125 gct gtt atc cgc gcc atg cct gtc tac aaaaaa gct gag cac gtc acg 432 Ala Val Ile Arg Ala Met Pro Val Tyr Lys LysAla Glu His Val Thr 130 135 140 gag gtg gtg aag cgg tgc ccc aac cat gagctg agc cgt gaa ttc aac 480 Glu Val Val Lys Arg Cys Pro Asn His Glu LeuSer Arg Glu Phe Asn 145 150 155 160 gag gga cag att gcc cct cct agt catttg att cga gta gag ggg aac 528 Glu Gly Gln Ile Ala Pro Pro Ser His LeuIle Arg Val Glu Gly Asn 165 170 175 agc cat gcc cag tat gta gaa gat cccatc aca gga aga cag agt gtg 576 Ser His Ala Gln Tyr Val Glu Asp Pro IleThr Gly Arg Gln Ser Val 180 185 190 ctg gta cct tat gag cca ccc cag gttggc act gaa ttc acg aca gtc 624 Leu Val Pro Tyr Glu Pro Pro Gln Val GlyThr Glu Phe Thr Thr Val 195 200 205 ttg tac aat ttc atg tgt aac agc agttgt gtt gga ggg atg aac cgc 672 Leu Tyr Asn Phe Met Cys Asn Ser Ser CysVal Gly Gly Met Asn Arg 210 215 220 cgt cca att tta atc att gtt act ctggaa acc aga gat ggg caa gtc 720 Arg Pro Ile Leu Ile Ile Val Thr Leu GluThr Arg Asp Gly Gln Val 225 230 235 240 ctg ggc cga cgc tgc ttt gag gcccgg atc tgt gct tgc cca gga aga 768 Leu Gly Arg Arg Cys Phe Glu Ala ArgIle Cys Ala Cys Pro Gly Arg 245 250 255 gac agg aag gcg gat gaa gat agcatc aga aag cag caa gtt tcg gac 816 Asp Arg Lys Ala Asp Glu Asp Ser IleArg Lys Gln Gln Val Ser Asp 260 265 270 agt aca aag aac ggt gat ggt acgaag cgc ccg ttt cgt cag aac aca 864 Ser Thr Lys Asn Gly Asp Gly Thr LysArg Pro Phe Arg Gln Asn Thr 275 280 285 cat ggt atc cag atg aca tcc atcaag aaa cga aga tcc cca gat gat 912 His Gly Ile Gln Met Thr Ser Ile LysLys Arg Arg Ser Pro Asp Asp 290 295 300 gaa ctg tta tac tta cca gtg aggggc cgt gag act tat gaa atg ctg 960 Glu Leu Leu Tyr Leu Pro Val Arg GlyArg Glu Thr Tyr Glu Met Leu 305 310 315 320 7 ttg aag atc aaa gag tccctg gaa ctc atg cag tac ctt cct cag cac 1008 Leu Lys Ile Lys Glu Ser LeuGlu Leu Met Gln Tyr Leu Pro Gln His 325 330 335 7 aca att gaa acg tacagg caa cag caa cag cag cag cac cag cac tta 1056 Thr Ile Glu Thr Tyr ArgGln Gln Gln Gln Gln Gln His Gln His Leu 340 345 350 7 ctt cag aaa cagacc tca ata cag tct cca tct tca tat ggt aac agc 1104 Leu Gln Lys Gln ThrSer Ile Gln Ser Pro Ser Ser Tyr Gly Asn Ser 355 360 365 7 tcc cca cctctg aac aaa atg aac agc atg aac aag ctg cct tct gtg 1152 Ser Pro Pro LeuAsn Lys Met Asn Ser Met Asn Lys Leu Pro Ser Val 370 375 380 7 agc cagctt atc aac cct cag cag cgc aac gcc ctc act cct aca acc 1200 Ser Gln LeuIle Asn Pro Gln Gln Arg Asn Ala Leu Thr Pro Thr Thr 385 390 395 400 7att cct gat ggc atg gga gcc aac att ccc atg atg ggc acc cac atg 1248 IlePro Asp Gly Met Gly Ala Asn Ile Pro Met Met Gly Thr His Met 405 410 4157 cca atg gct gga gac atg aat gga ctc agc ccc acc cag gca ctc cct 1296Pro Met Ala Gly Asp Met Asn Gly Leu Ser Pro Thr Gln Ala Leu Pro 420 425430 7 ccc cca ctc tcc atg cca tcc acc tcc cac tgc aca ccc cca cct ccg1344 Pro Pro Leu Ser Met Pro Ser Thr Ser His Cys Thr Pro Pro Pro Pro 435440 445 7 tat ccc aca gat tgc agc att gtc agt ttc tta gcg agg ttg ggctgt 1392 Tyr Pro Thr Asp Cys Ser Ile Val Ser Phe Leu Ala Arg Leu Gly Cys450 455 460 7 tca tca tgt ctg gac tat ttc acg acc cag ggg ctg acc accatc tat 1440 Ser Ser Cys Leu Asp Tyr Phe Thr Thr Gln Gly Leu Thr Thr IleTyr 465 470 475 480 7 cag att gag cat tac tcc atg gat gat ctg gca agtctg aaa atc cct 1488 Gln Ile Glu His Tyr Ser Met Asp Asp Leu Ala Ser LeuLys Ile Pro 485 490 495 7 gag caa ttt cga cat gcg atc tgg aag ggc atcctg gac cac cgg cag 1536 Glu Gln Phe Arg His Ala Ile Trp Lys Gly Ile LeuAsp His Arg Gln 500 505 510 7 ctc cac gaa ttc tcc tcc cct tct cat ctcctg cgg acc cca agc agt 1584 Leu His Glu Phe Ser Ser Pro Ser His Leu LeuArg Thr Pro Ser Ser 515 520 525 7 gcc tct aca gtc agt gtg ggc tcc agtgag acc cgg ggt gag cgt gtt 1632 Ala Ser Thr Val Ser Val Gly Ser Ser GluThr Arg Gly Glu Arg Val 530 535 540 7 att gat gct gtg cga ttc acc ctccgc cag acc atc tct ttc cca ccc 1680 Ile Asp Ala Val Arg Phe Thr Leu ArgGln Thr Ile Ser Phe Pro Pro 545 550 555 560 7 cga gat gag tgg aat gacttc aac ttt gac atg gat gct cgc cgc aat 1728 Arg Asp Glu Trp Asn Asp PheAsn Phe Asp Met Asp Ala Arg Arg Asn 565 570 575 7 aag caa cag cgc atcaaa gag gag ggg gag tga 1761 Lys Gln Gln Arg Ile Lys Glu Glu Gly Glu 580585 5 1386 DNA Homo sapiens CDS (1)..(1383) 5 atg ttg tac ctg gaa aacaat gcc cag act caa ttt agt gag cca cag 48 Met Leu Tyr Leu Glu Asn AsnAla Gln Thr Gln Phe Ser Glu Pro Gln 1 5 10 15 tac acg aac ctg ggg ctcctg aac agc atg gac cag cag att cag aac 96 Tyr Thr Asn Leu Gly Leu LeuAsn Ser Met Asp Gln Gln Ile Gln Asn 20 25 30 ggc tcc tcg tcc acc agt ccctat aac aca gac cac gcg cag aac agc 144 Gly Ser Ser Ser Thr Ser Pro TyrAsn Thr Asp His Ala Gln Asn Ser 35 40 45 gtc acg gcg ccc tcg ccc tac gcacag ccc agc tcc acc ttc gat gct 192 Val Thr Ala Pro Ser Pro Tyr Ala GlnPro Ser Ser Thr Phe Asp Ala 50 55 60 ctc tct cca tca ccc gcc atc ccc tccaac acc gac tac cca ggc ccg 240 Leu Ser Pro Ser Pro Ala Ile Pro Ser AsnThr Asp Tyr Pro Gly Pro 65 70 75 80 cac agt ttc gac gtg tcc ttc cag cagtcg agc acc gcc aag tcg gcc 288 His Ser Phe Asp Val Ser Phe Gln Gln SerSer Thr Ala Lys Ser Ala 85 90 95 acc tgg acg tat tcc act gaa ctg aag aaactc tac tgc caa att gca 336 Thr Trp Thr Tyr Ser Thr Glu Leu Lys Lys LeuTyr Cys Gln Ile Ala 100 105 110 aag aca tgc ccc atc cag atc aag gtg atgacc cca cct cct cag gga 384 Lys Thr Cys Pro Ile Gln Ile Lys Val Met ThrPro Pro Pro Gln Gly 115 120 125 gct gtt atc cgc gcc atg cct gtc tac aaaaaa gct gag cac gtc acg 432 Ala Val Ile Arg Ala Met Pro Val Tyr Lys LysAla Glu His Val Thr 130 135 140 gag gtg gtg aag cgg tgc ccc aac cat gagctg agc cgt gaa ttc aac 480 Glu Val Val Lys Arg Cys Pro Asn His Glu LeuSer Arg Glu Phe Asn 145 150 155 160 gag gga cag att gcc cct cct agt catttg att cga gta gag ggg aac 528 Glu Gly Gln Ile Ala Pro Pro Ser His LeuIle Arg Val Glu Gly Asn 165 170 175 agc cat gcc cag tat gta gaa gat cccatc aca gga aga cag agt gtg 576 Ser His Ala Gln Tyr Val Glu Asp Pro IleThr Gly Arg Gln Ser Val 180 185 190 ctg gta cct tat gag cca ccc cag gttggc act gaa ttc acg aca gtc 624 Leu Val Pro Tyr Glu Pro Pro Gln Val GlyThr Glu Phe Thr Thr Val 195 200 205 ttg tac aat ttc atg tgt aac agc agttgt gtt gga ggg atg aac cgc 672 Leu Tyr Asn Phe Met Cys Asn Ser Ser CysVal Gly Gly Met Asn Arg 210 215 220 cgt cca att tta atc att gtt act ctggaa acc aga gat ggg caa gtc 720 Arg Pro Ile Leu Ile Ile Val Thr Leu GluThr Arg Asp Gly Gln Val 225 230 235 240 ctg ggc cga cgc tgc ttt gag gcccgg atc tgt gct tgc cca gga aga 768 Leu Gly Arg Arg Cys Phe Glu Ala ArgIle Cys Ala Cys Pro Gly Arg 245 250 255 gac agg aag gcg gat gaa gat agcatc aga aag cag caa gtt tcg gac 816 Asp Arg Lys Ala Asp Glu Asp Ser IleArg Lys Gln Gln Val Ser Asp 260 265 270 agt aca aag aac ggt gat ggt acgaag cgc ccg ttt cgt cag aac aca 864 Ser Thr Lys Asn Gly Asp Gly Thr LysArg Pro Phe Arg Gln Asn Thr 275 280 285 cat ggt atc cag atg aca tcc atcaag aaa cga aga tcc cca gat gat 912 His Gly Ile Gln Met Thr Ser Ile LysLys Arg Arg Ser Pro Asp Asp 290 295 300 gaa ctg tta tac tta cca gtg aggggc cgt gag act tat gaa atg ctg 960 Glu Leu Leu Tyr Leu Pro Val Arg GlyArg Glu Thr Tyr Glu Met Leu 305 310 315 320 ttg aag atc aaa gag tcc ctggaa ctc atg cag tac ctt cct cag cac 1008 Leu Lys Ile Lys Glu Ser Leu GluLeu Met Gln Tyr Leu Pro Gln His 325 330 335 aca att gaa acg tac agg caacag caa cag cag cag cac cag cac tta 1056 Thr Ile Glu Thr Tyr Arg Gln GlnGln Gln Gln Gln His Gln His Leu 340 345 350 ctt cag aaa cag acc tca atacag tct cca tct tca tat ggt aac agc 1104 Leu Gln Lys Gln Thr Ser Ile GlnSer Pro Ser Ser Tyr Gly Asn Ser 355 360 365 tcc cca cct ctg aac aaa atgaac agc atg aac aag ctg cct tct gtg 1152 Ser Pro Pro Leu Asn Lys Met AsnSer Met Asn Lys Leu Pro Ser Val 370 375 380 agc cag ctt atc aac cct cagcag cgc aac gcc ctc act cct aca acc 1200 Ser Gln Leu Ile Asn Pro Gln GlnArg Asn Ala Leu Thr Pro Thr Thr 385 390 395 400 att cct gat ggc atg ggagcc aac att ccc atg atg ggc acc cac atg 1248 Ile Pro Asp Gly Met Gly AlaAsn Ile Pro Met Met Gly Thr His Met 405 410 415 cca atg gct gga gac atgaat gga ctc agc ccc acc cag gca ctc cct 1296 Pro Met Ala Gly Asp Met AsnGly Leu Ser Pro Thr Gln Ala Leu Pro 420 425 430 ccc cca ctc tcc atg ccatcc acc tcc cac tgc aca ccc cca cct ccg 1344 Pro Pro Leu Ser Met Pro SerThr Ser His Cys Thr Pro Pro Pro Pro 435 440 445 tat ccc aca gat tgc agcatt gtc agg atc tgg caa gtc tga 1386 Tyr Pro Thr Asp Cys Ser Ile Val ArgIle Trp Gln Val 450 455 460 6 1182 DNA Homo sapiens CDS (1)..(1179) 6atg ttg tac ctg gaa aac aat gcc cag act caa ttt agt gag cca cag 48 MetLeu Tyr Leu Glu Asn Asn Ala Gln Thr Gln Phe Ser Glu Pro Gln 1 5 10 15tac acg aac ctg ggg ctc ctg aac agc atg gac cag cag att cag aac 96 TyrThr Asn Leu Gly Leu Leu Asn Ser Met Asp Gln Gln Ile Gln Asn 20 25 30 ggctcc tcg tcc acc agt ccc tat aac aca gac cac gcg cag aac agc 144 Gly SerSer Ser Thr Ser Pro Tyr Asn Thr Asp His Ala Gln Asn Ser 35 40 45 gtc acggcg ccc tcg ccc tac gca cag ccc agc tcc acc ttc gat gct 192 Val Thr AlaPro Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe Asp Ala 50 55 60 ctc tct ccatca ccc gcc atc ccc tcc aac acc gac tac cca ggc ccg 240 Leu Ser Pro SerPro Ala Ile Pro Ser Asn Thr Asp Tyr Pro Gly Pro 65 70 75 80 cac agt ttcgac gtg tcc ttc cag cag tcg agc acc gcc aag tcg gcc 288 His Ser Phe AspVal Ser Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala 85 90 95 acc tgg acg tattcc act gaa ctg aag aaa ctc tac tgc caa att gca 336 Thr Trp Thr Tyr SerThr Glu Leu Lys Lys Leu Tyr Cys Gln Ile Ala 100 105 110 aag aca tgc cccatc cag atc aag gtg atg acc cca cct cct cag gga 384 Lys Thr Cys Pro IleGln Ile Lys Val Met Thr Pro Pro Pro Gln Gly 115 120 125 gct gtt atc cgcgcc atg cct gtc tac aaa aaa gct gag cac gtc acg 432 Ala Val Ile Arg AlaMet Pro Val Tyr Lys Lys Ala Glu His Val Thr 130 135 140 gag gtg gtg aagcgg tgc ccc aac cat gag ctg agc cgt gaa ttc aac 480 Glu Val Val Lys ArgCys Pro Asn His Glu Leu Ser Arg Glu Phe Asn 145 150 155 160 gag gga cagatt gcc cct cct agt cat ttg att cga gta gag ggg aac 528 Glu Gly Gln IleAla Pro Pro Ser His Leu Ile Arg Val Glu Gly Asn 165 170 175 agc cat gcccag tat gta gaa gat ccc atc aca gga aga cag agt gtg 576 Ser His Ala GlnTyr Val Glu Asp Pro Ile Thr Gly Arg Gln Ser Val 180 185 190 ctg gta ccttat gag cca ccc cag gtt ggc act gaa ttc acg aca gtc 624 Leu Val Pro TyrGlu Pro Pro Gln Val Gly Thr Glu Phe Thr Thr Val 195 200 205 ttg tac aatttc atg tgt aac agc agt tgt gtt gga ggg atg aac cgc 672 Leu Tyr Asn PheMet Cys Asn Ser Ser Cys Val Gly Gly Met Asn Arg 210 215 220 cgt cca atttta atc att gtt act ctg gaa acc aga gat ggg caa gtc 720 Arg Pro Ile LeuIle Ile Val Thr Leu Glu Thr Arg Asp Gly Gln Val 225 230 235 240 ctg ggccga cgc tgc ttt gag gcc cgg atc tgt gct tgc cca gga aga 768 Leu Gly ArgArg Cys Phe Glu Ala Arg Ile Cys Ala Cys Pro Gly Arg 245 250 255 gac aggaag gcg gat gaa gat agc atc aga aag cag caa gtt tcg gac 816 Asp Arg LysAla Asp Glu Asp Ser Ile Arg Lys Gln Gln Val Ser Asp 260 265 270 agt acaaag aac ggt gat ggt acg aag cgc ccg ttt cgt cag aac aca 864 Ser Thr LysAsn Gly Asp Gly Thr Lys Arg Pro Phe Arg Gln Asn Thr 275 280 285 cat ggtatc cag atg aca tcc atc aag aaa cga aga tcc cca gat gat 912 His Gly IleGln Met Thr Ser Ile Lys Lys Arg Arg Ser Pro Asp Asp 290 295 300 gaa ctgtta tac tta cca gtg agg ggc cgt gag act tat gaa atg ctg 960 Glu Leu LeuTyr Leu Pro Val Arg Gly Arg Glu Thr Tyr Glu Met Leu 305 310 315 320 ttgaag atc aaa gag tcc ctg gaa ctc atg cag tac ctt cct cag cac 1008 Leu LysIle Lys Glu Ser Leu Glu Leu Met Gln Tyr Leu Pro Gln His 325 330 335 acaatt gaa acg tac agg caa cag caa cag cag cag cac cag cac tta 1056 Thr IleGlu Thr Tyr Arg Gln Gln Gln Gln Gln Gln His Gln His Leu 340 345 350 cttcag aaa cat ctc ctt tca gcc tgc ttc agg aat gag ctt gtg gag 1104 Leu GlnLys His Leu Leu Ser Ala Cys Phe Arg Asn Glu Leu Val Glu 355 360 365 ccccgg aga gaa act cca aaa caa tct gac gtc ttc ttt aga cat tcc 1152 Pro ArgArg Glu Thr Pro Lys Gln Ser Asp Val Phe Phe Arg His Ser 370 375 380 aagccc cca aac cga tca gtg tac cca tag 1182 Lys Pro Pro Asn Arg Ser Val TyrPro 385 390 7 2043 DNA murine CDS (1)..(2040) 7 atg aat ttt gaa act tcacgg tgt gcc acc cta cag tac tgc ccc gac 48 Met Asn Phe Glu Thr Ser ArgCys Ala Thr Leu Gln Tyr Cys Pro Asp 1 5 10 15 cct tac atc cag cgt ttcata gaa acc cca gct cat ttc tcg tgg aaa 96 Pro Tyr Ile Gln Arg Phe IleGlu Thr Pro Ala His Phe Ser Trp Lys 20 25 30 gaa agt tat tac aga tct gccatg tcg cag agc acc cag aca agc gag 144 Glu Ser Tyr Tyr Arg Ser Ala MetSer Gln Ser Thr Gln Thr Ser Glu 35 40 45 ttc ctc agc cca gag gtc ttc cagcat atc tgg gat ttt ctg gaa cag 192 Phe Leu Ser Pro Glu Val Phe Gln HisIle Trp Asp Phe Leu Glu Gln 50 55 60 cct ata tgc tca gta cag ccc atc gagttg aac ttt gtg gat gaa cct 240 Pro Ile Cys Ser Val Gln Pro Ile Glu LeuAsn Phe Val Asp Glu Pro 65 70 75 80 tcc gaa aat ggt gca aca aac aag attgag att agc atg gat tgt atc 288 Ser Glu Asn Gly Ala Thr Asn Lys Ile GluIle Ser Met Asp Cys Ile 85 90 95 cgc atg caa gac tca gac ctc agt gac cccatg tgg cca cag tac acg 336 Arg Met Gln Asp Ser Asp Leu Ser Asp Pro MetTrp Pro Gln Tyr Thr 100 105 110 aac ctg ggg ctc ctg aac agc atg gac cagcag att cag aac ggc tcc 384 Asn Leu Gly Leu Leu Asn Ser Met Asp Gln GlnIle Gln Asn Gly Ser 115 120 125 tcg tcc acc agc ccc tac aac aca gac cacgca cag aat agc gtg acg 432 Ser Ser Thr Ser Pro Tyr Asn Thr Asp His AlaGln Asn Ser Val Thr 130 135 140 gcg ccc tcg ccc tat gca cag ccc agc tccacc ttt gat gcc ctc tct 480 Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser ThrPhe Asp Ala Leu Ser 145 150 155 160 cca tcc cct gcc att ccc tcc aac acagat tac ccg ggc cca cac agc 528 Pro Ser Pro Ala Ile Pro Ser Asn Thr AspTyr Pro Gly Pro His Ser 165 170 175 ttc gat gtg tcc ttc cag cag tca agcact gcc aag tca gcc acc tgg 576 Phe Asp Val Ser Phe Gln Gln Ser Ser ThrAla Lys Ser Ala Thr Trp 180 185 190 acg tat tcc acc gaa ctg aag aag ctgtac tgc cag att gcg aag aca 624 Thr Tyr Ser Thr Glu Leu Lys Lys Leu TyrCys Gln Ile Ala Lys Thr 195 200 205 tgc ccc atc cag atc aag gtg atg acccca ccc cca cag ggc gct gtt 672 Cys Pro Ile Gln Ile Lys Val Met Thr ProPro Pro Gln Gly Ala Val 210 215 220 atc cgt gcc atg cct gtc tac aag aaagct gag cat gtc acc gag gtt 720 Ile Arg Ala Met Pro Val Tyr Lys Lys AlaGlu His Val Thr Glu Val 225 230 235 240 gtg aaa cga tgc cct aac cat gagctg agc cgt gag ttc aat gag gga 768 Val Lys Arg Cys Pro Asn His Glu LeuSer Arg Glu Phe Asn Glu Gly 245 250 255 cag att gcc cct ccc agt cat ctgatt cga gta gaa ggg aac agc cat 816 Gln Ile Ala Pro Pro Ser His Leu IleArg Val Glu Gly Asn Ser His 260 265 270 gcc cag tat gta gaa gat cct atcacg gga agg cag agc gtg ctg gtc 864 Ala Gln Tyr Val Glu Asp Pro Ile ThrGly Arg Gln Ser Val Leu Val 275 280 285 cct tat gag cca cca cag gtt ggcact gaa ttc aca aca gtc ctg tac 912 Pro Tyr Glu Pro Pro Gln Val Gly ThrGlu Phe Thr Thr Val Leu Tyr 290 295 300 aat ttc atg tgt aac agc agc tgcgtc gga gga atg aac aga cgt cca 960 Asn Phe Met Cys Asn Ser Ser Cys ValGly Gly Met Asn Arg Arg Pro 305 310 315 320 att tta atc atc gtt act ctggaa acc aga gat ggg caa gtc ctg ggc 1008 Ile Leu Ile Ile Val Thr Leu GluThr Arg Asp Gly Gln Val Leu Gly 325 330 335 cga cgg tgc ttt gag gcc cggatc tgt gct tgc cca gga aga gac cgg 1056 Arg Arg Cys Phe Glu Ala Arg IleCys Ala Cys Pro Gly Arg Asp Arg 340 345 350 aag gca gat gaa gac agc atcaga aag cag caa gta tcg gac agc gca 1104 Lys Ala Asp Glu Asp Ser Ile ArgLys Gln Gln Val Ser Asp Ser Ala 355 360 365 aag aac ggc gat ggt acg aagcgc cct ttc cgt cag aat aca cac gga 1152 Lys Asn Gly Asp Gly Thr Lys ArgPro Phe Arg Gln Asn Thr His Gly 370 375 380 atc cag atg act tcc atc aagaaa cgg aga tcc cca gat gat gag ctg 1200 Ile Gln Met Thr Ser Ile Lys LysArg Arg Ser Pro Asp Asp Glu Leu 385 390 395 400 ctg tac cta cca gtg agaggt cgt gag acg tac gag atg ttg ctg aag 1248 Leu Tyr Leu Pro Val Arg GlyArg Glu Thr Tyr Glu Met Leu Leu Lys 405 410 415 atc aaa gag tca ctg gagctc atg cag tac ctc cct cag cac acg atc 1296 Ile Lys Glu Ser Leu Glu LeuMet Gln Tyr Leu Pro Gln His Thr Ile 420 425 430 gaa acg tac agg cag cagcag cag cag cag cac cag cac cta ctt cag 1344 Glu Thr Tyr Arg Gln Gln GlnGln Gln Gln His Gln His Leu Leu Gln 435 440 445 aaa cag acc tcg atg cagtct cag tct tca tat ggc aac agt tcc cca 1392 Lys Gln Thr Ser Met Gln SerGln Ser Ser Tyr Gly Asn Ser Ser Pro 450 455 460 cct ctg aac aaa atg aacagc atg aac aag ctg cct tcc gtg agc cag 1440 Pro Leu Asn Lys Met Asn SerMet Asn Lys Leu Pro Ser Val Ser Gln 465 470 475 480 ctt atc aac cca cagcag cgc aat gcc ctc act ccc acc acc atg cct 1488 Leu Ile Asn Pro Gln GlnArg Asn Ala Leu Thr Pro Thr Thr Met Pro 485 490 495 gag ggc atg gga gccaac att cct atg atg ggc act cac atg cca atg 1536 Glu Gly Met Gly Ala AsnIle Pro Met Met Gly Thr His Met Pro Met 500 505 510 gct gga gac atg aatgga ctc agc cct acc caa gct ctc cct cct cca 1584 Ala Gly Asp Met Asn GlyLeu Ser Pro Thr Gln Ala Leu Pro Pro Pro 515 520 525 ctc tcc atg ccc tccacc tcc cac tgc acc cca cca ccg ccc tac ccc 1632 Leu Ser Met Pro Ser ThrSer His Cys Thr Pro Pro Pro Pro Tyr Pro 530 535 540 aca gac tgc agc attgtc agt ttc tta gca agg ttg ggc tgc tca tca 1680 Thr Asp Cys Ser Ile ValSer Phe Leu Ala Arg Leu Gly Cys Ser Ser 545 550 555 560 tgc ctg gac tatttc acg acc cag ggg ctg acc acc atc tat cag att 1728 Cys Leu Asp Tyr PheThr Thr Gln Gly Leu Thr Thr Ile Tyr Gln Ile 565 570 575 gag cat tac tccatg gat gat ttg gca agt ctg aag atc cct gaa cag 1776 Glu His Tyr Ser MetAsp Asp Leu Ala Ser Leu Lys Ile Pro Glu Gln 580 585 590 ttc cga cat gccatc tgg aag ggc atc ctg gac cac agg cag ctg cac 1824 Phe Arg His Ala IleTrp Lys Gly Ile Leu Asp His Arg Gln Leu His 595 600 605 gac ttc tcc tcacct cct cat ctc ctg agg acc cca agt ggt gcc tct 1872 Asp Phe Ser Ser ProPro His Leu Leu Arg Thr Pro Ser Gly Ala Ser 610 615 620 acc gtc agt gtgggc tcc agt gag acc cgt ggt gaa cgt gtg atc gat 1920 Thr Val Ser Val GlySer Ser Glu Thr Arg Gly Glu Arg Val Ile Asp 625 630 635 640 gcc gtg cgcttt acc ctc cgc cag acc atc tct ttt cca ccc cgt gac 1968 Ala Val Arg PheThr Leu Arg Gln Thr Ile Ser Phe Pro Pro Arg Asp 645 650 655 gag tgg aatgat ttc aac ttt gac atg gat tct cgt cgc aac aag cag 2016 Glu Trp Asn AspPhe Asn Phe Asp Met Asp Ser Arg Arg Asn Lys Gln 660 665 670 cag cgt atcaaa gag gaa gga gaa tga 2043 Gln Arg Ile Lys Glu Glu Gly Glu 675 680 81668 DNA murine CDS (1)..(1665) 8 atg aat ttt gaa act tca cgg tgt gccacc cta cag tac tgc ccc gac 48 Met Asn Phe Glu Thr Ser Arg Cys Ala ThrLeu Gln Tyr Cys Pro Asp 1 5 10 15 cct tac atc cag cgt ttc ata gaa acccca gct cat ttc tcg tgg aaa 96 Pro Tyr Ile Gln Arg Phe Ile Glu Thr ProAla His Phe Ser Trp Lys 20 25 30 gaa agt tat tac aga tct gcc atg tcg cagagc acc cag aca agc gag 144 Glu Ser Tyr Tyr Arg Ser Ala Met Ser Gln SerThr Gln Thr Ser Glu 35 40 45 ttc ctc agc cca gag gtc ttc cag cat atc tgggat ttt ctg gaa cag 192 Phe Leu Ser Pro Glu Val Phe Gln His Ile Trp AspPhe Leu Glu Gln 50 55 60 cct ata tgc tca gta cag ccc atc gag ttg aac tttgtg gat gaa cct 240 Pro Ile Cys Ser Val Gln Pro Ile Glu Leu Asn Phe ValAsp Glu Pro 65 70 75 80 tcc gaa aat ggt gca aca aac aag att gag att agcatg gat tgt atc 288 Ser Glu Asn Gly Ala Thr Asn Lys Ile Glu Ile Ser MetAsp Cys Ile 85 90 95 cgc atg caa gac tca gac ctc agt gac ccc atg tgg ccacag tac acg 336 Arg Met Gln Asp Ser Asp Leu Ser Asp Pro Met Trp Pro GlnTyr Thr 100 105 110 aac ctg ggg ctc ctg aac agc atg gac cag cag att cagaac ggc tcc 384 Asn Leu Gly Leu Leu Asn Ser Met Asp Gln Gln Ile Gln AsnGly Ser 115 120 125 tcg tcc acc agc ccc tac aac aca gac cac gca cag aatagc gtg acg 432 Ser Ser Thr Ser Pro Tyr Asn Thr Asp His Ala Gln Asn SerVal Thr 130 135 140 gcg ccc tcg ccc tat gca cag ccc agc tcc acc ttt gatgcc ctc tct 480 Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe Asp AlaLeu Ser 145 150 155 160 cca tcc cct gcc att ccc tcc aac aca gat tac ccgggc cca cac agc 528 Pro Ser Pro Ala Ile Pro Ser Asn Thr Asp Tyr Pro GlyPro His Ser 165 170 175 ttc gat gtg tcc ttc cag cag tca agc act gcc aagtca gcc acc tgg 576 Phe Asp Val Ser Phe Gln Gln Ser Ser Thr Ala Lys SerAla Thr Trp 180 185 190 acg tat tcc acc gaa ctg aag aag ctg tac tgc cagatt gcg aag aca 624 Thr Tyr Ser Thr Glu Leu Lys Lys Leu Tyr Cys Gln IleAla Lys Thr 195 200 205 tgc ccc atc cag atc aag gtg atg acc cca ccc ccacag ggc gct gtt 672 Cys Pro Ile Gln Ile Lys Val Met Thr Pro Pro Pro GlnGly Ala Val 210 215 220 atc cgt gcc atg cct gtc tac aag aaa gct gag catgtc acc gag gtt 720 Ile Arg Ala Met Pro Val Tyr Lys Lys Ala Glu His ValThr Glu Val 225 230 235 240 gtg aaa cga tgc cct aac cat gag ctg agc cgtgag ttc aat gag gga 768 Val Lys Arg Cys Pro Asn His Glu Leu Ser Arg GluPhe Asn Glu Gly 245 250 255 cag att gcc cct ccc agt cat ctg att cga gtagaa ggg aac agc cat 816 Gln Ile Ala Pro Pro Ser His Leu Ile Arg Val GluGly Asn Ser His 260 265 270 gcc cag tat gta gaa gat cct atc acg gga aggcag agc gtg ctg gtc 864 Ala Gln Tyr Val Glu Asp Pro Ile Thr Gly Arg GlnSer Val Leu Val 275 280 285 cct tat gag cca cca cag gtt ggc act gaa ttcaca aca gtc ctg tac 912 Pro Tyr Glu Pro Pro Gln Val Gly Thr Glu Phe ThrThr Val Leu Tyr 290 295 300 aat ttc atg tgt aac agc agc tgc gtc gga ggaatg aac aga cgt cca 960 Asn Phe Met Cys Asn Ser Ser Cys Val Gly Gly MetAsn Arg Arg Pro 305 310 315 320 att tta atc atc gtt act ctg gaa acc agagat ggg caa gtc ctg ggc 1008 Ile Leu Ile Ile Val Thr Leu Glu Thr Arg AspGly Gln Val Leu Gly 325 330 335 cga cgg tgc ttt gag gcc cgg atc tgt gcttgc cca gga aga gac cgg 1056 Arg Arg Cys Phe Glu Ala Arg Ile Cys Ala CysPro Gly Arg Asp Arg 340 345 350 aag gca gat gaa gac agc atc aga aag cagcaa gta tcg gac agc gca 1104 Lys Ala Asp Glu Asp Ser Ile Arg Lys Gln GlnVal Ser Asp Ser Ala 355 360 365 aag aac ggc gat ggt acg aag cgc cct ttccgt cag aat aca cac gga 1152 Lys Asn Gly Asp Gly Thr Lys Arg Pro Phe ArgGln Asn Thr His Gly 370 375 380 atc cag atg act tcc atc aag aaa cgg agatcc cca gat gat gag ctg 1200 Ile Gln Met Thr Ser Ile Lys Lys Arg Arg SerPro Asp Asp Glu Leu 385 390 395 400 ctg tac cta cca gtg aga ggt cgt gagacg tac gag atg ttg ctg aag 1248 Leu Tyr Leu Pro Val Arg Gly Arg Glu ThrTyr Glu Met Leu Leu Lys 405 410 415 atc aaa gag tca ctg gag ctc atg cagtac ctc cct cag cac acg atc 1296 Ile Lys Glu Ser Leu Glu Leu Met Gln TyrLeu Pro Gln His Thr Ile 420 425 430 gaa acg tac agg cag cag cag cag cagcag cac cag cac cta ctt cag 1344 Glu Thr Tyr Arg Gln Gln Gln Gln Gln GlnHis Gln His Leu Leu Gln 435 440 445 aaa cag acc tcg atg cag tct cag tcttca tat ggc aac agt tcc cca 1392 Lys Gln Thr Ser Met Gln Ser Gln Ser SerTyr Gly Asn Ser Ser Pro 450 455 460 cct ctg aac aaa atg aac agc atg aacaag ctg cct tcc gtg agc cag 1440 Pro Leu Asn Lys Met Asn Ser Met Asn LysLeu Pro Ser Val Ser Gln 465 470 475 480 ctt atc aac cca cag cag cgc aatgcc ctc act ccc acc acc atg cct 1488 Leu Ile Asn Pro Gln Gln Arg Asn AlaLeu Thr Pro Thr Thr Met Pro 485 490 495 gag ggc atg gga gcc aac att cctatg atg ggc act cac atg cca atg 1536 Glu Gly Met Gly Ala Asn Ile Pro MetMet Gly Thr His Met Pro Met 500 505 510 gct gga gac atg aat gga ctc agccct acc caa gct ctc cct cct cca 1584 Ala Gly Asp Met Asn Gly Leu Ser ProThr Gln Ala Leu Pro Pro Pro 515 520 525 ctc tcc atg ccc tcc acc tcc cactgc acc cca cca ccg ccc tac ccc 1632 Leu Ser Met Pro Ser Thr Ser His CysThr Pro Pro Pro Pro Tyr Pro 530 535 540 aca gac tgc agc att gtc agg atttgg caa gtc tga 1668 Thr Asp Cys Ser Ile Val Arg Ile Trp Gln Val 545 550555 9 1452 DNA murine CDS (1)..(1449) 9 atg aat ttt gaa act tca cgg tgtgcc acc cta cag tac tgc ccc gac 48 Met Asn Phe Glu Thr Ser Arg Cys AlaThr Leu Gln Tyr Cys Pro Asp 1 5 10 15 cct tac atc cag cgt ttc ata gaaacc cca gct cat ttc tcg tgg aaa 96 Pro Tyr Ile Gln Arg Phe Ile Glu ThrPro Ala His Phe Ser Trp Lys 20 25 30 gaa agt tat tac aga tct gcc atg tcgcag agc acc cag aca agc gag 144 Glu Ser Tyr Tyr Arg Ser Ala Met Ser GlnSer Thr Gln Thr Ser Glu 35 40 45 ttc ctc agc cca gag gtc ttc cag cat atctgg gat ttt ctg gaa cag 192 Phe Leu Ser Pro Glu Val Phe Gln His Ile TrpAsp Phe Leu Glu Gln 50 55 60 cct ata tgc tca gta cag ccc atc gag ttg aacttt gtg gat gaa cct 240 Pro Ile Cys Ser Val Gln Pro Ile Glu Leu Asn PheVal Asp Glu Pro 65 70 75 80 tcc gaa aat ggt gca aca aac aag att gag attagc atg gat tgt atc 288 Ser Glu Asn Gly Ala Thr Asn Lys Ile Glu Ile SerMet Asp Cys Ile 85 90 95 cgc atg caa gac tca gac ctc agt gac ccc atg tggcca cag tac acg 336 Arg Met Gln Asp Ser Asp Leu Ser Asp Pro Met Trp ProGln Tyr Thr 100 105 110 aac ctg ggg ctc ctg aac agc atg gac cag cag attcag aac ggc tcc 384 Asn Leu Gly Leu Leu Asn Ser Met Asp Gln Gln Ile GlnAsn Gly Ser 115 120 125 tcg tcc acc agc ccc tac aac aca gac cac gca cagaat agc gtg acg 432 Ser Ser Thr Ser Pro Tyr Asn Thr Asp His Ala Gln AsnSer Val Thr 130 135 140 gcg ccc tcg ccc tat gca cag ccc agc tcc acc tttgat gcc ctc tct 480 Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe AspAla Leu Ser 145 150 155 160 cca tcc cct gcc att ccc tcc aac aca gat tacccg ggc cca cac agc 528 Pro Ser Pro Ala Ile Pro Ser Asn Thr Asp Tyr ProGly Pro His Ser 165 170 175 ttc gat gtg tcc ttc cag cag tca agc act gccaag tca gcc acc tgg 576 Phe Asp Val Ser Phe Gln Gln Ser Ser Thr Ala LysSer Ala Thr Trp 180 185 190 acg tat tcc acc gaa ctg aag aag ctg tac tgccag att gcg aag aca 624 Thr Tyr Ser Thr Glu Leu Lys Lys Leu Tyr Cys GlnIle Ala Lys Thr 195 200 205 tgc ccc atc cag atc aag gtg atg acc cca ccccca cag ggc gct gtt 672 Cys Pro Ile Gln Ile Lys Val Met Thr Pro Pro ProGln Gly Ala Val 210 215 220 atc cgt gcc atg cct gtc tac aag aaa gct gagcat gtc acc gag gtt 720 Ile Arg Ala Met Pro Val Tyr Lys Lys Ala Glu HisVal Thr Glu Val 225 230 235 240 gtg aaa cga tgc cct aac cat gag ctg agccgt gag ttc aat gag gga 768 Val Lys Arg Cys Pro Asn His Glu Leu Ser ArgGlu Phe Asn Glu Gly 245 250 255 cag att gcc cct ccc agt cat ctg att cgagta gaa ggg aac agc cat 816 Gln Ile Ala Pro Pro Ser His Leu Ile Arg ValGlu Gly Asn Ser His 260 265 270 gcc cag tat gta gaa gat cct atc acg ggaagg cag agc gtg ctg gtc 864 Ala Gln Tyr Val Glu Asp Pro Ile Thr Gly ArgGln Ser Val Leu Val 275 280 285 cct tat gag cca cca cag gtt ggc act gaattc aca aca gtc ctg tac 912 Pro Tyr Glu Pro Pro Gln Val Gly Thr Glu PheThr Thr Val Leu Tyr 290 295 300 aat ttc atg tgt aac agc agc tgc gtc ggagga atg aac aga cgt cca 960 Asn Phe Met Cys Asn Ser Ser Cys Val Gly GlyMet Asn Arg Arg Pro 305 310 315 320 att tta atc atc gtt act ctg gaa accaga gat ggg caa gtc ctg ggc 1008 Ile Leu Ile Ile Val Thr Leu Glu Thr ArgAsp Gly Gln Val Leu Gly 325 330 335 cga cgg tgc ttt gag gcc cgg atc tgtgct tgc cca gga aga gac cgg 1056 Arg Arg Cys Phe Glu Ala Arg Ile Cys AlaCys Pro Gly Arg Asp Arg 340 345 350 aag gca gat gaa gac agc atc aga aagcag caa gta tcg gac agc gca 1104 Lys Ala Asp Glu Asp Ser Ile Arg Lys GlnGln Val Ser Asp Ser Ala 355 360 365 aag aac ggc gat gct ttc cgt cag aataca cac gga atc cag atg act 1152 Lys Asn Gly Asp Ala Phe Arg Gln Asn ThrHis Gly Ile Gln Met Thr 370 375 380 tcc atc aag aaa cgg aga tcc cca gatgat gag ctg ctg tac cta cca 1200 Ser Ile Lys Lys Arg Arg Ser Pro Asp AspGlu Leu Leu Tyr Leu Pro 385 390 395 400 gtg aga ggt cgt gag acg tac gagatg ttg ctg aag atc aaa gag tca 1248 Val Arg Gly Arg Glu Thr Tyr Glu MetLeu Leu Lys Ile Lys Glu Ser 405 410 415 ctg gag ctc atg cag tac ctc cctcag cac acg atc gaa acg tac agg 1296 Leu Glu Leu Met Gln Tyr Leu Pro GlnHis Thr Ile Glu Thr Tyr Arg 420 425 430 cag cag cag cag cag cag cac cagcac cta ctt cag aaa cat ctc ctt 1344 Gln Gln Gln Gln Gln Gln His Gln HisLeu Leu Gln Lys His Leu Leu 435 440 445 tca gcc tgc ttc agg aat gag cttgtg gag ccc cgg gga gaa gct ccg 1392 Ser Ala Cys Phe Arg Asn Glu Leu ValGlu Pro Arg Gly Glu Ala Pro 450 455 460 aca cag tct gac gtc ttc ttt agacat tcc aac ccc cca aac cac tcc 1440 Thr Gln Ser Asp Val Phe Phe Arg HisSer Asn Pro Pro Asn His Ser 465 470 475 480 gtg tac cca tag 1452 Val TyrPro 10 1761 DNA murine CDS (1)..(1758) 10 atg ttg tac ctg gaa aac aatgcc cag act caa ttt agt gag cca cag 48 Met Leu Tyr Leu Glu Asn Asn AlaGln Thr Gln Phe Ser Glu Pro Gln 1 5 10 15 tac acg aac ctg ggg ctc ctgaac agc atg gac cag cag att cag aac 96 Tyr Thr Asn Leu Gly Leu Leu AsnSer Met Asp Gln Gln Ile Gln Asn 20 25 30 ggc tcc tcg tcc acc agc ccc tacaac aca gac cac gca cag aat agc 144 Gly Ser Ser Ser Thr Ser Pro Tyr AsnThr Asp His Ala Gln Asn Ser 35 40 45 gtg acg gcg ccc tcg ccc tat gca cagccc agc tcc acc ttt gat gcc 192 Val Thr Ala Pro Ser Pro Tyr Ala Gln ProSer Ser Thr Phe Asp Ala 50 55 60 ctc tct cca tcc cct gcc att ccc tcc aacaca gat tac ccg ggc cca 240 Leu Ser Pro Ser Pro Ala Ile Pro Ser Asn ThrAsp Tyr Pro Gly Pro 65 70 75 80 cac agc ttc gat gtg tcc ttc cag cag tcaagc act gcc aag tca gcc 288 His Ser Phe Asp Val Ser Phe Gln Gln Ser SerThr Ala Lys Ser Ala 85 90 95 acc tgg acg tat tcc acc gaa ctg aag aag ctgtac tgc cag att gcg 336 Thr Trp Thr Tyr Ser Thr Glu Leu Lys Lys Leu TyrCys Gln Ile Ala 100 105 110 aag aca tgc ccc atc cag atc aag gtg atg acccca ccc cca cag ggc 384 Lys Thr Cys Pro Ile Gln Ile Lys Val Met Thr ProPro Pro Gln Gly 115 120 125 gct gtt atc cgt gcc atg cct gtc tac aag aaagct gag cat gtc acc 432 Ala Val Ile Arg Ala Met Pro Val Tyr Lys Lys AlaGlu His Val Thr 130 135 140 gag gtt gtg aaa cga tgc cct aac cat gag ctgagc cgt gag ttc aat 480 Glu Val Val Lys Arg Cys Pro Asn His Glu Leu SerArg Glu Phe Asn 145 150 155 160 gag gga cag att gcc cct ccc agt cat ctgatt cga gta gaa ggg aac 528 Glu Gly Gln Ile Ala Pro Pro Ser His Leu IleArg Val Glu Gly Asn 165 170 175 agc cat gcc cag tat gta gaa gat cct atcacg gga agg cag agc gtg 576 Ser His Ala Gln Tyr Val Glu Asp Pro Ile ThrGly Arg Gln Ser Val 180 185 190 ctg gtc cct tat gag cca cca cag gtt ggcact gaa ttc aca aca gtc 624 Leu Val Pro Tyr Glu Pro Pro Gln Val Gly ThrGlu Phe Thr Thr Val 195 200 205 ctg tac aat ttc atg tgt aac agc agc tgcgtc gga gga atg aac aga 672 Leu Tyr Asn Phe Met Cys Asn Ser Ser Cys ValGly Gly Met Asn Arg 210 215 220 cgt cca att tta atc atc gtt act ctg gaaacc aga gat ggg caa gtc 720 Arg Pro Ile Leu Ile Ile Val Thr Leu Glu ThrArg Asp Gly Gln Val 225 230 235 240 ctg ggc cga cgg tgc ttt gag gcc cggatc tgt gct tgc cca gga aga 768 Leu Gly Arg Arg Cys Phe Glu Ala Arg IleCys Ala Cys Pro Gly Arg 245 250 255 gac cgg aag gca gat gaa gac agc atcaga aag cag caa gta tcg gac 816 Asp Arg Lys Ala Asp Glu Asp Ser Ile ArgLys Gln Gln Val Ser Asp 260 265 270 agc gca aag aac ggc gat ggt acg aagcgc cct ttc cgt cag aat aca 864 Ser Ala Lys Asn Gly Asp Gly Thr Lys ArgPro Phe Arg Gln Asn Thr 275 280 285 cac gga atc cag atg act tcc atc aagaaa cgg aga tcc cca gat gat 912 His Gly Ile Gln Met Thr Ser Ile Lys LysArg Arg Ser Pro Asp Asp 290 295 300 gag ctg ctg tac cta cca gtg aga ggtcgt gag acg tac gag atg ttg 960 Glu Leu Leu Tyr Leu Pro Val Arg Gly ArgGlu Thr Tyr Glu Met Leu 305 310 315 320 ctg aag atc aaa gag tca ctg gagctc atg cag tac ctc cct cag cac 1008 Leu Lys Ile Lys Glu Ser Leu Glu LeuMet Gln Tyr Leu Pro Gln His 325 330 335 acg atc gaa acg tac agg cag cagcag cag cag cag cac cag cac cta 1056 Thr Ile Glu Thr Tyr Arg Gln Gln GlnGln Gln Gln His Gln His Leu 340 345 350 ctt cag aaa cag acc tcg atg cagtct cag tct tca tat ggc aac agt 1104 Leu Gln Lys Gln Thr Ser Met Gln SerGln Ser Ser Tyr Gly Asn Ser 355 360 365 tcc cca cct ctg aac aaa atg aacagc atg aac aag ctg cct tcc gtg 1152 Ser Pro Pro Leu Asn Lys Met Asn SerMet Asn Lys Leu Pro Ser Val 370 375 380 agc cag ctt atc aac cca cag cagcgc aat gcc ctc act ccc acc acc 1200 Ser Gln Leu Ile Asn Pro Gln Gln ArgAsn Ala Leu Thr Pro Thr Thr 385 390 395 400 atg cct gag ggc atg gga gccaac att cct atg atg ggc act cac atg 1248 Met Pro Glu Gly Met Gly Ala AsnIle Pro Met Met Gly Thr His Met 405 410 415 cca atg gct gga gac atg aatgga ctc agc cct acc caa gct ctc cct 1296 Pro Met Ala Gly Asp Met Asn GlyLeu Ser Pro Thr Gln Ala Leu Pro 420 425 430 cct cca ctc tcc atg ccc tccacc tcc cac tgc acc cca cca ccg ccc 1344 Pro Pro Leu Ser Met Pro Ser ThrSer His Cys Thr Pro Pro Pro Pro 435 440 445 tac ccc aca gac tgc agc attgtc agt ttc tta gca agg ttg ggc tgc 1392 Tyr Pro Thr Asp Cys Ser Ile ValSer Phe Leu Ala Arg Leu Gly Cys 450 455 460 tca tca tgc ctg gac tat ttcacg acc cag ggg ctg acc acc atc tat 1440 Ser Ser Cys Leu Asp Tyr Phe ThrThr Gln Gly Leu Thr Thr Ile Tyr 465 470 475 480 cag att gag cat tac tccatg gat gat ttg gca agt ctg aag atc cct 1488 Gln Ile Glu His Tyr Ser MetAsp Asp Leu Ala Ser Leu Lys Ile Pro 485 490 495 gaa cag ttc cga cat gccatc tgg aag ggc atc ctg gac cac agg cag 1536 Glu Gln Phe Arg His Ala IleTrp Lys Gly Ile Leu Asp His Arg Gln 500 505 510 ctg cac gac ttc tcc tcacct cct cat ctc ctg agg acc cca agt ggt 1584 Leu His Asp Phe Ser Ser ProPro His Leu Leu Arg Thr Pro Ser Gly 515 520 525 gcc tct acc gtc agt gtgggc tcc agt gag acc cgt ggt gaa cgt gtg 1632 Ala Ser Thr Val Ser Val GlySer Ser Glu Thr Arg Gly Glu Arg Val 530 535 540 atc gat gcc gtg cgc tttacc ctc cgc cag acc atc tct ttt cca ccc 1680 Ile Asp Ala Val Arg Phe ThrLeu Arg Gln Thr Ile Ser Phe Pro Pro 545 550 555 560 cgt gac gag tgg aatgat ttc aac ttt gac atg gat tct cgt cgc aac 1728 Arg Asp Glu Trp Asn AspPhe Asn Phe Asp Met Asp Ser Arg Arg Asn 565 570 575 aag cag cag cgt atcaaa gag gaa gga gaa tga 1761 Lys Gln Gln Arg Ile Lys Glu Glu Gly Glu 580585 11 1386 DNA murine CDS (1)..(1383) 11 atg ttg tac ctg gaa aac aatgcc cag act caa ttt agt gag cca cag 48 Met Leu Tyr Leu Glu Asn Asn AlaGln Thr Gln Phe Ser Glu Pro Gln 1 5 10 15 tac acg aac ctg ggg ctc ctgaac agc atg gac cag cag att cag aac 96 Tyr Thr Asn Leu Gly Leu Leu AsnSer Met Asp Gln Gln Ile Gln Asn 20 25 30 ggc tcc tcg tcc acc agc ccc tacaac aca gac cac gca cag aat agc 144 Gly Ser Ser Ser Thr Ser Pro Tyr AsnThr Asp His Ala Gln Asn Ser 35 40 45 gtg acg gcg ccc tcg ccc tat gca cagccc agc tcc acc ttt gat gcc 192 Val Thr Ala Pro Ser Pro Tyr Ala Gln ProSer Ser Thr Phe Asp Ala 50 55 60 ctc tct cca tcc cct gcc att ccc tcc aacaca gat tac ccg ggc cca 240 Leu Ser Pro Ser Pro Ala Ile Pro Ser Asn ThrAsp Tyr Pro Gly Pro 65 70 75 80 cac agc ttc gat gtg tcc ttc cag cag tcaagc act gcc aag tca gcc 288 His Ser Phe Asp Val Ser Phe Gln Gln Ser SerThr Ala Lys Ser Ala 85 90 95 acc tgg acg tat tcc acc gaa ctg aag aag ctgtac tgc cag att gcg 336 Thr Trp Thr Tyr Ser Thr Glu Leu Lys Lys Leu TyrCys Gln Ile Ala 100 105 110 aag aca tgc ccc atc cag atc aag gtg atg acccca ccc cca cag ggc 384 Lys Thr Cys Pro Ile Gln Ile Lys Val Met Thr ProPro Pro Gln Gly 115 120 125 gct gtt atc cgt gcc atg cct gtc tac aag aaagct gag cat gtc acc 432 Ala Val Ile Arg Ala Met Pro Val Tyr Lys Lys AlaGlu His Val Thr 130 135 140 gag gtt gtg aaa cga tgc cct aac cat gag ctgagc cgt gag ttc aat 480 Glu Val Val Lys Arg Cys Pro Asn His Glu Leu SerArg Glu Phe Asn 145 150 155 160 gag gga cag att gcc cct ccc agt cat ctgatt cga gta gaa ggg aac 528 Glu Gly Gln Ile Ala Pro Pro Ser His Leu IleArg Val Glu Gly Asn 165 170 175 agc cat gcc cag tat gta gaa gat cct atcacg gga agg cag agc gtg 576 Ser His Ala Gln Tyr Val Glu Asp Pro Ile ThrGly Arg Gln Ser Val 180 185 190 ctg gtc cct tat gag cca cca cag gtt ggcact gaa ttc aca aca gtc 624 Leu Val Pro Tyr Glu Pro Pro Gln Val Gly ThrGlu Phe Thr Thr Val 195 200 205 ctg tac aat ttc atg tgt aac agc agc tgcgtc gga gga atg aac aga 672 Leu Tyr Asn Phe Met Cys Asn Ser Ser Cys ValGly Gly Met Asn Arg 210 215 220 cgt cca att tta atc atc gtt act ctg gaaacc aga gat ggg caa gtc 720 Arg Pro Ile Leu Ile Ile Val Thr Leu Glu ThrArg Asp Gly Gln Val 225 230 235 240 ctg ggc cga cgg tgc ttt gag gcc cggatc tgt gct tgc cca gga aga 768 Leu Gly Arg Arg Cys Phe Glu Ala Arg IleCys Ala Cys Pro Gly Arg 245 250 255 gac cgg aag gca gat gaa gac agc atcaga aag cag caa gta tcg gac 816 Asp Arg Lys Ala Asp Glu Asp Ser Ile ArgLys Gln Gln Val Ser Asp 260 265 270 agc gca aag aac ggc gat ggt acg aagcgc cct ttc cgt cag aat aca 864 Ser Ala Lys Asn Gly Asp Gly Thr Lys ArgPro Phe Arg Gln Asn Thr 275 280 285 cac gga atc cag atg act tcc atc aagaaa cgg aga tcc cca gat gat 912 His Gly Ile Gln Met Thr Ser Ile Lys LysArg Arg Ser Pro Asp Asp 290 295 300 gag ctg ctg tac cta cca gtg aga ggtcgt gag acg tac gag atg ttg 960 Glu Leu Leu Tyr Leu Pro Val Arg Gly ArgGlu Thr Tyr Glu Met Leu 305 310 315 320 ctg aag atc aaa gag tca ctg gagctc atg cag tac ctc cct cag cac 1008 Leu Lys Ile Lys Glu Ser Leu Glu LeuMet Gln Tyr Leu Pro Gln His 325 330 335 acg atc gaa acg tac agg cag cagcag cag cag cag cac cag cac cta 1056 Thr Ile Glu Thr Tyr Arg Gln Gln GlnGln Gln Gln His Gln His Leu 340 345 350 ctt cag aaa cag acc tcg atg cagtct cag tct tca tat ggc aac agt 1104 Leu Gln Lys Gln Thr Ser Met Gln SerGln Ser Ser Tyr Gly Asn Ser 355 360 365 tcc cca cct ctg aac aaa atg aacagc atg aac aag ctg cct tcc gtg 1152 Ser Pro Pro Leu Asn Lys Met Asn SerMet Asn Lys Leu Pro Ser Val 370 375 380 agc cag ctt atc aac cca cag cagcgc aat gcc ctc act ccc acc acc 1200 Ser Gln Leu Ile Asn Pro Gln Gln ArgAsn Ala Leu Thr Pro Thr Thr 385 390 395 400 atg cct gag ggc atg gga gccaac att cct atg atg ggc act cac atg 1248 Met Pro Glu Gly Met Gly Ala AsnIle Pro Met Met Gly Thr His Met 405 410 415 cca atg gct gga gac atg aatgga ctc agc cct acc caa gct ctc cct 1296 Pro Met Ala Gly Asp Met Asn GlyLeu Ser Pro Thr Gln Ala Leu Pro 420 425 430 cct cca ctc tcc atg ccc tccacc tcc cac tgc acc cca cca ccg ccc 1344 Pro Pro Leu Ser Met Pro Ser ThrSer His Cys Thr Pro Pro Pro Pro 435 440 445 tac ccc aca gac tgc agc attgtc agg att tgg caa gtc tga 1386 Tyr Pro Thr Asp Cys Ser Ile Val Arg IleTrp Gln Val 450 455 460 12 1170 DNA murine CDS (1)..(1167) 12 atg ttgtac ctg gaa aac aat gcc cag act caa ttt agt gag cca cag 48 Met Leu TyrLeu Glu Asn Asn Ala Gln Thr Gln Phe Ser Glu Pro Gln 1 5 10 15 tac acgaac ctg ggg ctc ctg aac agc atg gac cag cag att cag aac 96 Tyr Thr AsnLeu Gly Leu Leu Asn Ser Met Asp Gln Gln Ile Gln Asn 20 25 30 ggc tcc tcgtcc acc agc ccc tac aac aca gac cac gca cag aat agc 144 Gly Ser Ser SerThr Ser Pro Tyr Asn Thr Asp His Ala Gln Asn Ser 35 40 45 gtg acg gcg ccctcg ccc tat gca cag ccc agc tcc acc ttt gat gcc 192 Val Thr Ala Pro SerPro Tyr Ala Gln Pro Ser Ser Thr Phe Asp Ala 50 55 60 ctc tct cca tcc cctgcc att ccc tcc aac aca gat tac ccg ggc cca 240 Leu Ser Pro Ser Pro AlaIle Pro Ser Asn Thr Asp Tyr Pro Gly Pro 65 70 75 80 cac agc ttc gat gtgtcc ttc cag cag tca agc act gcc aag tca gcc 288 His Ser Phe Asp Val SerPhe Gln Gln Ser Ser Thr Ala Lys Ser Ala 85 90 95 acc tgg acg tat tcc accgaa ctg aag aag ctg tac tgc cag att gcg 336 Thr Trp Thr Tyr Ser Thr GluLeu Lys Lys Leu Tyr Cys Gln Ile Ala 100 105 110 aag aca tgc ccc atc cagatc aag gtg atg acc cca ccc cca cag ggc 384 Lys Thr Cys Pro Ile Gln IleLys Val Met Thr Pro Pro Pro Gln Gly 115 120 125 gct gtt atc cgt gcc atgcct gtc tac aag aaa gct gag cat gtc acc 432 Ala Val Ile Arg Ala Met ProVal Tyr Lys Lys Ala Glu His Val Thr 130 135 140 gag gtt gtg aaa cga tgccct aac cat gag ctg agc cgt gag ttc aat 480 Glu Val Val Lys Arg Cys ProAsn His Glu Leu Ser Arg Glu Phe Asn 145 150 155 160 gag gga cag att gcccct ccc agt cat ctg att cga gta gaa ggg aac 528 Glu Gly Gln Ile Ala ProPro Ser His Leu Ile Arg Val Glu Gly Asn 165 170 175 agc cat gcc cag tatgta gaa gat cct atc acg gga agg cag agc gtg 576 Ser His Ala Gln Tyr ValGlu Asp Pro Ile Thr Gly Arg Gln Ser Val 180 185 190 ctg gtc cct tat gagcca cca cag gtt ggc act gaa ttc aca aca gtc 624 Leu Val Pro Tyr Glu ProPro Gln Val Gly Thr Glu Phe Thr Thr Val 195 200 205 ctg tac aat ttc atgtgt aac agc agc tgc gtc gga gga atg aac aga 672 Leu Tyr Asn Phe Met CysAsn Ser Ser Cys Val Gly Gly Met Asn Arg 210 215 220 cgt cca att tta atcatc gtt act ctg gaa acc aga gat ggg caa gtc 720 Arg Pro Ile Leu Ile IleVal Thr Leu Glu Thr Arg Asp Gly Gln Val 225 230 235 240 ctg ggc cga cggtgc ttt gag gcc cgg atc tgt gct tgc cca gga aga 768 Leu Gly Arg Arg CysPhe Glu Ala Arg Ile Cys Ala Cys Pro Gly Arg 245 250 255 gac cgg aag gcagat gaa gac agc atc aga aag cag caa gta tcg gac 816 Asp Arg Lys Ala AspGlu Asp Ser Ile Arg Lys Gln Gln Val Ser Asp 260 265 270 agc gca aag aacggc gat gct ttc cgt cag aat aca cac gga atc cag 864 Ser Ala Lys Asn GlyAsp Ala Phe Arg Gln Asn Thr His Gly Ile Gln 275 280 285 atg act tcc atcaag aaa cgg aga tcc cca gat gat gag ctg ctg tac 912 Met Thr Ser Ile LysLys Arg Arg Ser Pro Asp Asp Glu Leu Leu Tyr 290 295 300 cta cca gtg agaggt cgt gag acg tac gag atg ttg ctg aag atc aaa 960 Leu Pro Val Arg GlyArg Glu Thr Tyr Glu Met Leu Leu Lys Ile Lys 305 310 315 320 gag tca ctggag ctc atg cag tac ctc cct cag cac acg atc gaa acg 1008 Glu Ser Leu GluLeu Met Gln Tyr Leu Pro Gln His Thr Ile Glu Thr 325 330 335 tac agg cagcag cag cag cag cag cac cag cac cta ctt cag aaa cat 1056 Tyr Arg Gln GlnGln Gln Gln Gln His Gln His Leu Leu Gln Lys His 340 345 350 ctc ctt tcagcc tgc ttc agg aat gag ctt gtg gag ccc cgg gga gaa 1104 Leu Leu Ser AlaCys Phe Arg Asn Glu Leu Val Glu Pro Arg Gly Glu 355 360 365 gct ccg acacag tct gac gtc ttc ttt aga cat tcc aac ccc cca aac 1152 Ala Pro Thr GlnSer Asp Val Phe Phe Arg His Ser Asn Pro Pro Asn 370 375 380 cac tcc gtgtac cca tag 1170 His Ser Val Tyr Pro 385 13 641 PRT Homo sapiens 13 MetSer Gln Ser Thr Gln Thr Asn Glu Phe Leu Ser Pro Glu Val Phe 1 5 10 15Gln His Ile Trp Asp Phe Leu Glu Gln Pro Ile Cys Ser Val Gln Pro 20 25 30Ile Asp Leu Asn Phe Val Asp Glu Pro Ser Glu Asp Gly Ala Thr Asn 35 40 45Lys Ile Glu Ile Ser Met Asp Cys Ile Arg Met Gln Asp Ser Asp Leu 50 55 60Ser Asp Pro Met Trp Pro Gln Tyr Thr Asn Leu Gly Leu Leu Asn Ser 65 70 7580 Met Asp Gln Gln Ile Gln Asn Gly Ser Ser Ser Thr Ser Pro Tyr Asn 85 9095 Thr Asp His Ala Gln Asn Ser Val Thr Ala Pro Ser Pro Tyr Ala Gln 100105 110 Pro Ser Ser Thr Phe Asp Ala Leu Ser Pro Ser Pro Ala Ile Pro Ser115 120 125 Asn Thr Asp Tyr Pro Gly Pro His Ser Phe Asp Val Ser Phe GlnGln 130 135 140 Ser Ser Thr Ala Lys Ser Ala Thr Trp Thr Tyr Ser Thr GluLeu Lys 145 150 155 160 Lys Leu Tyr Cys Gln Ile Ala Lys Thr Cys Pro IleGln Ile Lys Val 165 170 175 Met Thr Pro Pro Pro Gln Gly Ala Val Ile ArgAla Met Pro Val Tyr 180 185 190 Lys Lys Ala Glu His Val Thr Glu Val ValLys Arg Cys Pro Asn His 195 200 205 Glu Leu Ser Arg Glu Phe Asn Glu GlyGln Ile Ala Pro Pro Ser His 210 215 220 Leu Ile Arg Val Glu Gly Asn SerHis Ala Gln Tyr Val Glu Asp Pro 225 230 235 240 Ile Thr Gly Arg Gln SerVal Leu Val Pro Tyr Glu Pro Pro Gln Val 245 250 255 Gly Thr Glu Phe ThrThr Val Leu Tyr Asn Phe Met Cys Asn Ser Ser 260 265 270 Cys Val Gly GlyMet Asn Arg Arg Pro Ile Leu Ile Ile Val Thr Leu 275 280 285 Glu Thr ArgAsp Gly Gln Val Leu Gly Arg Arg Cys Phe Glu Ala Arg 290 295 300 Ile CysAla Cys Pro Gly Arg Asp Arg Lys Ala Asp Glu Asp Ser Ile 305 310 315 320Arg Lys Gln Gln Val Ser Asp Ser Thr Lys Asn Gly Asp Gly Thr Lys 325 330335 Arg Pro Phe Arg Gln Asn Thr His Gly Ile Gln Met Thr Ser Ile Lys 340345 350 Lys Arg Arg Ser Pro Asp Asp Glu Leu Leu Tyr Leu Pro Val Arg Gly355 360 365 Arg Glu Thr Tyr Glu Met Leu Leu Lys Ile Lys Glu Ser Leu GluLeu 370 375 380 Met Gln Tyr Leu Pro Gln His Thr Ile Glu Thr Tyr Arg GlnGln Gln 385 390 395 400 Gln Gln Gln His Gln His Leu Leu Gln Lys Gln ThrSer Ile Gln Ser 405 410 415 Pro Ser Ser Tyr Gly Asn Ser Ser Pro Pro LeuAsn Lys Met Asn Ser 420 425 430 Met Asn Lys Leu Pro Ser Val Ser Gln LeuIle Asn Pro Gln Gln Arg 435 440 445 Asn Ala Leu Thr Pro Thr Thr Ile ProAsp Gly Met Gly Ala Asn Ile 450 455 460 Pro Met Met Gly Thr His Met ProMet Ala Gly Asp Met Asn Gly Leu 465 470 475 480 Ser Pro Thr Gln Ala LeuPro Pro Pro Leu Ser Met Pro Ser Thr Ser 485 490 495 His Cys Thr Pro ProPro Pro Tyr Pro Thr Asp Cys Ser Ile Val Ser 500 505 510 Phe Leu Ala ArgLeu Gly Cys Ser Ser Cys Leu Asp Tyr Phe Thr Thr 515 520 525 Gln Gly LeuThr Thr Ile Tyr Gln Ile Glu His Tyr Ser Met Asp Asp 530 535 540 Leu AlaSer Leu Lys Ile Pro Glu Gln Phe Arg His Ala Ile Trp Lys 545 550 555 560Gly Ile Leu Asp His Arg Gln Leu His Glu Phe Ser Ser Pro Ser His 565 570575 Leu Leu Arg Thr Pro Ser Ser Ala Ser Thr Val Ser Val Gly Ser Ser 580585 590 Glu Thr Arg Gly Glu Arg Val Ile Asp Ala Val Arg Phe Thr Leu Arg595 600 605 Gln Thr Ile Ser Phe Pro Pro Arg Asp Glu Trp Asn Asp Phe AsnPhe 610 615 620 Asp Met Asp Ala Arg Arg Asn Lys Gln Gln Arg Ile Lys GluGlu Gly 625 630 635 640 Glu 14 516 PRT Homo sapiens 14 Met Ser Gln SerThr Gln Thr Asn Glu Phe Leu Ser Pro Glu Val Phe 1 5 10 15 Gln His IleTrp Asp Phe Leu Glu Gln Pro Ile Cys Ser Val Gln Pro 20 25 30 Ile Asp LeuAsn Phe Val Asp Glu Pro Ser Glu Asp Gly Ala Thr Asn 35 40 45 Lys Ile GluIle Ser Met Asp Cys Ile Arg Met Gln Asp Ser Asp Leu 50 55 60 Ser Asp ProMet Trp Pro Gln Tyr Thr Asn Leu Gly Leu Leu Asn Ser 65 70 75 80 Met AspGln Gln Ile Gln Asn Gly Ser Ser Ser Thr Ser Pro Tyr Asn 85 90 95 Thr AspHis Ala Gln Asn Ser Val Thr Ala Pro Ser Pro Tyr Ala Gln 100 105 110 ProSer Ser Thr Phe Asp Ala Leu Ser Pro Ser Pro Ala Ile Pro Ser 115 120 125Asn Thr Asp Tyr Pro Gly Pro His Ser Phe Asp Val Ser Phe Gln Gln 130 135140 Ser Ser Thr Ala Lys Ser Ala Thr Trp Thr Tyr Ser Thr Glu Leu Lys 145150 155 160 Lys Leu Tyr Cys Gln Ile Ala Lys Thr Cys Pro Ile Gln Ile LysVal 165 170 175 Met Thr Pro Pro Pro Gln Gly Ala Val Ile Arg Ala Met ProVal Tyr 180 185 190 Lys Lys Ala Glu His Val Thr Glu Val Val Lys Arg CysPro Asn His 195 200 205 Glu Leu Ser Arg Glu Phe Asn Glu Gly Gln Ile AlaPro Pro Ser His 210 215 220 Leu Ile Arg Val Glu Gly Asn Ser His Ala GlnTyr Val Glu Asp Pro 225 230 235 240 Ile Thr Gly Arg Gln Ser Val Leu ValPro Tyr Glu Pro Pro Gln Val 245 250 255 Gly Thr Glu Phe Thr Thr Val LeuTyr Asn Phe Met Cys Asn Ser Ser 260 265 270 Cys Val Gly Gly Met Asn ArgArg Pro Ile Leu Ile Ile Val Thr Leu 275 280 285 Glu Thr Arg Asp Gly GlnVal Leu Gly Arg Arg Cys Phe Glu Ala Arg 290 295 300 Ile Cys Ala Cys ProGly Arg Asp Arg Lys Ala Asp Glu Asp Ser Ile 305 310 315 320 Arg Lys GlnGln Val Ser Asp Ser Thr Lys Asn Gly Asp Gly Thr Lys 325 330 335 Arg ProPhe Arg Gln Asn Thr His Gly Ile Gln Met Thr Ser Ile Lys 340 345 350 LysArg Arg Ser Pro Asp Asp Glu Leu Leu Tyr Leu Pro Val Arg Gly 355 360 365Arg Glu Thr Tyr Glu Met Leu Leu Lys Ile Lys Glu Ser Leu Glu Leu 370 375380 Met Gln Tyr Leu Pro Gln His Thr Ile Glu Thr Tyr Arg Gln Gln Gln 385390 395 400 Gln Gln Gln His Gln His Leu Leu Gln Lys Gln Thr Ser Ile GlnSer 405 410 415 Pro Ser Ser Tyr Gly Asn Ser Ser Pro Pro Leu Asn Lys MetAsn Ser 420 425 430 Met Asn Lys Leu Pro Ser Val Ser Gln Leu Ile Asn ProGln Gln Arg 435 440 445 Asn Ala Leu Thr Pro Thr Thr Ile Pro Asp Gly MetGly Ala Asn Ile 450 455 460 Pro Met Met Gly Thr His Met Pro Met Ala GlyAsp Met Asn Gly Leu 465 470 475 480 Ser Pro Thr Gln Ala Leu Pro Pro ProLeu Ser Met Pro Ser Thr Ser 485 490 495 His Cys Thr Pro Pro Pro Pro TyrPro Thr Asp Cys Ser Ile Val Arg 500 505 510 Ile Trp Gln Val 515 15 448PRT Homo sapiens 15 Met Ser Gln Ser Thr Gln Thr Asn Glu Phe Leu Ser ProGlu Val Phe 1 5 10 15 Gln His Ile Trp Asp Phe Leu Glu Gln Pro Ile CysSer Val Gln Pro 20 25 30 Ile Asp Leu Asn Phe Val Asp Glu Pro Ser Glu AspGly Ala Thr Asn 35 40 45 Lys Ile Glu Ile Ser Met Asp Cys Ile Arg Met GlnAsp Ser Asp Leu 50 55 60 Ser Asp Pro Met Trp Pro Gln Tyr Thr Asn Leu GlyLeu Leu Asn Ser 65 70 75 80 Met Asp Gln Gln Ile Gln Asn Gly Ser Ser SerThr Ser Pro Tyr Asn 85 90 95 Thr Asp His Ala Gln Asn Ser Val Thr Ala ProSer Pro Tyr Ala Gln 100 105 110 Pro Ser Ser Thr Phe Asp Ala Leu Ser ProSer Pro Ala Ile Pro Ser 115 120 125 Asn Thr Asp Tyr Pro Gly Pro His SerPhe Asp Val Ser Phe Gln Gln 130 135 140 Ser Ser Thr Ala Lys Ser Ala ThrTrp Thr Tyr Ser Thr Glu Leu Lys 145 150 155 160 Lys Leu Tyr Cys Gln IleAla Lys Thr Cys Pro Ile Gln Ile Lys Val 165 170 175 Met Thr Pro Pro ProGln Gly Ala Val Ile Arg Ala Met Pro Val Tyr 180 185 190 Lys Lys Ala GluHis Val Thr Glu Val Val Lys Arg Cys Pro Asn His 195 200 205 Glu Leu SerArg Glu Phe Asn Glu Gly Gln Ile Ala Pro Pro Ser His 210 215 220 Leu IleArg Val Glu Gly Asn Ser His Ala Gln Tyr Val Glu Asp Pro 225 230 235 240Ile Thr Gly Arg Gln Ser Val Leu Val Pro Tyr Glu Pro Pro Gln Val 245 250255 Gly Thr Glu Phe Thr Thr Val Leu Tyr Asn Phe Met Cys Asn Ser Ser 260265 270 Cys Val Gly Gly Met Asn Arg Arg Pro Ile Leu Ile Ile Val Thr Leu275 280 285 Glu Thr Arg Asp Gly Gln Val Leu Gly Arg Arg Cys Phe Glu AlaArg 290 295 300 Ile Cys Ala Cys Pro Gly Arg Asp Arg Lys Ala Asp Glu AspSer Ile 305 310 315 320 Arg Lys Gln Gln Val Ser Asp Ser Thr Lys Asn GlyAsp Gly Thr Lys 325 330 335 Arg Pro Phe Arg Gln Asn Thr His Gly Ile GlnMet Thr Ser Ile Lys 340 345 350 Lys Arg Arg Ser Pro Asp Asp Glu Leu LeuTyr Leu Pro Val Arg Gly 355 360 365 Arg Glu Thr Tyr Glu Met Leu Leu LysIle Lys Glu Ser Leu Glu Leu 370 375 380 Met Gln Tyr Leu Pro Gln His ThrIle Glu Thr Tyr Arg Gln Gln Gln 385 390 395 400 Gln Gln Gln His Gln HisLeu Leu Gln Lys His Leu Leu Ser Ala Cys 405 410 415 Phe Arg Asn Glu LeuVal Glu Pro Arg Arg Glu Thr Pro Lys Gln Ser 420 425 430 Asp Val Phe PheArg His Ser Lys Pro Pro Asn Arg Ser Val Tyr Pro 435 440 445 16 586 PRTHomo sapiens 16 Met Leu Tyr Leu Glu Asn Asn Ala Gln Thr Gln Phe Ser GluPro Gln 1 5 10 15 Tyr Thr Asn Leu Gly Leu Leu Asn Ser Met Asp Gln GlnIle Gln Asn 20 25 30 Gly Ser Ser Ser Thr Ser Pro Tyr Asn Thr Asp His AlaGln Asn Ser 35 40 45 Val Thr Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser ThrPhe Asp Ala 50 55 60 Leu Ser Pro Ser Pro Ala Ile Pro Ser Asn Thr Asp TyrPro Gly Pro 65 70 75 80 His Ser Phe Asp Val Ser Phe Gln Gln Ser Ser ThrAla Lys Ser Ala 85 90 95 Thr Trp Thr Tyr Ser Thr Glu Leu Lys Lys Leu TyrCys Gln Ile Ala 100 105 110 Lys Thr Cys Pro Ile Gln Ile Lys Val Met ThrPro Pro Pro Gln Gly 115 120 125 Ala Val Ile Arg Ala Met Pro Val Tyr LysLys Ala Glu His Val Thr 130 135 140 Glu Val Val Lys Arg Cys Pro Asn HisGlu Leu Ser Arg Glu Phe Asn 145 150 155 160 Glu Gly Gln Ile Ala Pro ProSer His Leu Ile Arg Val Glu Gly Asn 165 170 175 Ser His Ala Gln Tyr ValGlu Asp Pro Ile Thr Gly Arg Gln Ser Val 180 185 190 Leu Val Pro Tyr GluPro Pro Gln Val Gly Thr Glu Phe Thr Thr Val 195 200 205 Leu Tyr Asn PheMet Cys Asn Ser Ser Cys Val Gly Gly Met Asn Arg 210 215 220 Arg Pro IleLeu Ile Ile Val Thr Leu Glu Thr Arg Asp Gly Gln Val 225 230 235 240 LeuGly Arg Arg Cys Phe Glu Ala Arg Ile Cys Ala Cys Pro Gly Arg 245 250 255Asp Arg Lys Ala Asp Glu Asp Ser Ile Arg Lys Gln Gln Val Ser Asp 260 265270 Ser Thr Lys Asn Gly Asp Gly Thr Lys Arg Pro Phe Arg Gln Asn Thr 275280 285 His Gly Ile Gln Met Thr Ser Ile Lys Lys Arg Arg Ser Pro Asp Asp290 295 300 Glu Leu Leu Tyr Leu Pro Val Arg Gly Arg Glu Thr Tyr Glu MetLeu 305 310 315 320 Leu Lys Ile Lys Glu Ser Leu Glu Leu Met Gln Tyr LeuPro Gln His 325 330 335 Thr Ile Glu Thr Tyr Arg Gln Gln Gln Gln Gln GlnHis Gln His Leu 340 345 350 Leu Gln Lys Gln Thr Ser Ile Gln Ser Pro SerSer Tyr Gly Asn Ser 355 360 365 Ser Pro Pro Leu Asn Lys Met Asn Ser MetAsn Lys Leu Pro Ser Val 370 375 380 Ser Gln Leu Ile Asn Pro Gln Gln ArgAsn Ala Leu Thr Pro Thr Thr 385 390 395 400 Ile Pro Asp Gly Met Gly AlaAsn Ile Pro Met Met Gly Thr His Met 405 410 415 Pro Met Ala Gly Asp MetAsn Gly Leu Ser Pro Thr Gln Ala Leu Pro 420 425 430 Pro Pro Leu Ser MetPro Ser Thr Ser His Cys Thr Pro Pro Pro Pro 435 440 445 Tyr Pro Thr AspCys Ser Ile Val Ser Phe Leu Ala Arg Leu Gly Cys 450 455 460 Ser Ser CysLeu Asp Tyr Phe Thr Thr Gln Gly Leu Thr Thr Ile Tyr 465 470 475 480 GlnIle Glu His Tyr Ser Met Asp Asp Leu Ala Ser Leu Lys Ile Pro 485 490 495Glu Gln Phe Arg His Ala Ile Trp Lys Gly Ile Leu Asp His Arg Gln 500 505510 Leu His Glu Phe Ser Ser Pro Ser His Leu Leu Arg Thr Pro Ser Ser 515520 525 Ala Ser Thr Val Ser Val Gly Ser Ser Glu Thr Arg Gly Glu Arg Val530 535 540 Ile Asp Ala Val Arg Phe Thr Leu Arg Gln Thr Ile Ser Phe ProPro 545 550 555 560 Arg Asp Glu Trp Asn Asp Phe Asn Phe Asp Met Asp AlaArg Arg Asn 565 570 575 Lys Gln Gln Arg Ile Lys Glu Glu Gly Glu 580 58517 461 PRT Homo sapiens 17 Met Leu Tyr Leu Glu Asn Asn Ala Gln Thr GlnPhe Ser Glu Pro Gln 1 5 10 15 Tyr Thr Asn Leu Gly Leu Leu Asn Ser MetAsp Gln Gln Ile Gln Asn 20 25 30 Gly Ser Ser Ser Thr Ser Pro Tyr Asn ThrAsp His Ala Gln Asn Ser 35 40 45 Val Thr Ala Pro Ser Pro Tyr Ala Gln ProSer Ser Thr Phe Asp Ala 50 55 60 Leu Ser Pro Ser Pro Ala Ile Pro Ser AsnThr Asp Tyr Pro Gly Pro 65 70 75 80 His Ser Phe Asp Val Ser Phe Gln GlnSer Ser Thr Ala Lys Ser Ala 85 90 95 Thr Trp Thr Tyr Ser Thr Glu Leu LysLys Leu Tyr Cys Gln Ile Ala 100 105 110 Lys Thr Cys Pro Ile Gln Ile LysVal Met Thr Pro Pro Pro Gln Gly 115 120 125 Ala Val Ile Arg Ala Met ProVal Tyr Lys Lys Ala Glu His Val Thr 130 135 140 Glu Val Val Lys Arg CysPro Asn His Glu Leu Ser Arg Glu Phe Asn 145 150 155 160 Glu Gly Gln IleAla Pro Pro Ser His Leu Ile Arg Val Glu Gly Asn 165 170 175 Ser His AlaGln Tyr Val Glu Asp Pro Ile Thr Gly Arg Gln Ser Val 180 185 190 Leu ValPro Tyr Glu Pro Pro Gln Val Gly Thr Glu Phe Thr Thr Val 195 200 205 LeuTyr Asn Phe Met Cys Asn Ser Ser Cys Val Gly Gly Met Asn Arg 210 215 220Arg Pro Ile Leu Ile Ile Val Thr Leu Glu Thr Arg Asp Gly Gln Val 225 230235 240 Leu Gly Arg Arg Cys Phe Glu Ala Arg Ile Cys Ala Cys Pro Gly Arg245 250 255 Asp Arg Lys Ala Asp Glu Asp Ser Ile Arg Lys Gln Gln Val SerAsp 260 265 270 Ser Thr Lys Asn Gly Asp Gly Thr Lys Arg Pro Phe Arg GlnAsn Thr 275 280 285 His Gly Ile Gln Met Thr Ser Ile Lys Lys Arg Arg SerPro Asp Asp 290 295 300 Glu Leu Leu Tyr Leu Pro Val Arg Gly Arg Glu ThrTyr Glu Met Leu 305 310 315 320 Leu Lys Ile Lys Glu Ser Leu Glu Leu MetGln Tyr Leu Pro Gln His 325 330 335 Thr Ile Glu Thr Tyr Arg Gln Gln GlnGln Gln Gln His Gln His Leu 340 345 350 Leu Gln Lys Gln Thr Ser Ile GlnSer Pro Ser Ser Tyr Gly Asn Ser 355 360 365 Ser Pro Pro Leu Asn Lys MetAsn Ser Met Asn Lys Leu Pro Ser Val 370 375 380 Ser Gln Leu Ile Asn ProGln Gln Arg Asn Ala Leu Thr Pro Thr Thr 385 390 395 400 Ile Pro Asp GlyMet Gly Ala Asn Ile Pro Met Met Gly Thr His Met 405 410 415 Pro Met AlaGly Asp Met Asn Gly Leu Ser Pro Thr Gln Ala Leu Pro 420 425 430 Pro ProLeu Ser Met Pro Ser Thr Ser His Cys Thr Pro Pro Pro Pro 435 440 445 TyrPro Thr Asp Cys Ser Ile Val Arg Ile Trp Gln Val 450 455 460 18 393 PRTHomo sapiens 18 Met Leu Tyr Leu Glu Asn Asn Ala Gln Thr Gln Phe Ser GluPro Gln 1 5 10 15 Tyr Thr Asn Leu Gly Leu Leu Asn Ser Met Asp Gln GlnIle Gln Asn 20 25 30 Gly Ser Ser Ser Thr Ser Pro Tyr Asn Thr Asp His AlaGln Asn Ser 35 40 45 Val Thr Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser ThrPhe Asp Ala 50 55 60 Leu Ser Pro Ser Pro Ala Ile Pro Ser Asn Thr Asp TyrPro Gly Pro 65 70 75 80 His Ser Phe Asp Val Ser Phe Gln Gln Ser Ser ThrAla Lys Ser Ala 85 90 95 Thr Trp Thr Tyr Ser Thr Glu Leu Lys Lys Leu TyrCys Gln Ile Ala 100 105 110 Lys Thr Cys Pro Ile Gln Ile Lys Val Met ThrPro Pro Pro Gln Gly 115 120 125 Ala Val Ile Arg Ala Met Pro Val Tyr LysLys Ala Glu His Val Thr 130 135 140 Glu Val Val Lys Arg Cys Pro Asn HisGlu Leu Ser Arg Glu Phe Asn 145 150 155 160 Glu Gly Gln Ile Ala Pro ProSer His Leu Ile Arg Val Glu Gly Asn 165 170 175 Ser His Ala Gln Tyr ValGlu Asp Pro Ile Thr Gly Arg Gln Ser Val 180 185 190 Leu Val Pro Tyr GluPro Pro Gln Val Gly Thr Glu Phe Thr Thr Val 195 200 205 Leu Tyr Asn PheMet Cys Asn Ser Ser Cys Val Gly Gly Met Asn Arg 210 215 220 Arg Pro IleLeu Ile Ile Val Thr Leu Glu Thr Arg Asp Gly Gln Val 225 230 235 240 LeuGly Arg Arg Cys Phe Glu Ala Arg Ile Cys Ala Cys Pro Gly Arg 245 250 255Asp Arg Lys Ala Asp Glu Asp Ser Ile Arg Lys Gln Gln Val Ser Asp 260 265270 Ser Thr Lys Asn Gly Asp Gly Thr Lys Arg Pro Phe Arg Gln Asn Thr 275280 285 His Gly Ile Gln Met Thr Ser Ile Lys Lys Arg Arg Ser Pro Asp Asp290 295 300 Glu Leu Leu Tyr Leu Pro Val Arg Gly Arg Glu Thr Tyr Glu MetLeu 305 310 315 320 Leu Lys Ile Lys Glu Ser Leu Glu Leu Met Gln Tyr LeuPro Gln His 325 330 335 Thr Ile Glu Thr Tyr Arg Gln Gln Gln Gln Gln GlnHis Gln His Leu 340 345 350 Leu Gln Lys His Leu Leu Ser Ala Cys Phe ArgAsn Glu Leu Val Glu 355 360 365 Pro Arg Arg Glu Thr Pro Lys Gln Ser AspVal Phe Phe Arg His Ser 370 375 380 Lys Pro Pro Asn Arg Ser Val Tyr Pro385 390 19 680 PRT murine 19 Met Asn Phe Glu Thr Ser Arg Cys Ala Thr LeuGln Tyr Cys Pro Asp 1 5 10 15 Pro Tyr Ile Gln Arg Phe Ile Glu Thr ProAla His Phe Ser Trp Lys 20 25 30 Glu Ser Tyr Tyr Arg Ser Ala Met Ser GlnSer Thr Gln Thr Ser Glu 35 40 45 Phe Leu Ser Pro Glu Val Phe Gln His IleTrp Asp Phe Leu Glu Gln 50 55 60 Pro Ile Cys Ser Val Gln Pro Ile Glu LeuAsn Phe Val Asp Glu Pro 65 70 75 80 Ser Glu Asn Gly Ala Thr Asn Lys IleGlu Ile Ser Met Asp Cys Ile 85 90 95 Arg Met Gln Asp Ser Asp Leu Ser AspPro Met Trp Pro Gln Tyr Thr 100 105 110 Asn Leu Gly Leu Leu Asn Ser MetAsp Gln Gln Ile Gln Asn Gly Ser 115 120 125 Ser Ser Thr Ser Pro Tyr AsnThr Asp His Ala Gln Asn Ser Val Thr 130 135 140 Ala Pro Ser Pro Tyr AlaGln Pro Ser Ser Thr Phe Asp Ala Leu Ser 145 150 155 160 Pro Ser Pro AlaIle Pro Ser Asn Thr Asp Tyr Pro Gly Pro His Ser 165 170 175 Phe Asp ValSer Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala Thr Trp 180 185 190 Thr TyrSer Thr Glu Leu Lys Lys Leu Tyr Cys Gln Ile Ala Lys Thr 195 200 205 CysPro Ile Gln Ile Lys Val Met Thr Pro Pro Pro Gln Gly Ala Val 210 215 220Ile Arg Ala Met Pro Val Tyr Lys Lys Ala Glu His Val Thr Glu Val 225 230235 240 Val Lys Arg Cys Pro Asn His Glu Leu Ser Arg Glu Phe Asn Glu Gly245 250 255 Gln Ile Ala Pro Pro Ser His Leu Ile Arg Val Glu Gly Asn SerHis 260 265 270 Ala Gln Tyr Val Glu Asp Pro Ile Thr Gly Arg Gln Ser ValLeu Val 275 280 285 Pro Tyr Glu Pro Pro Gln Val Gly Thr Glu Phe Thr ThrVal Leu Tyr 290 295 300 Asn Phe Met Cys Asn Ser Ser Cys Val Gly Gly MetAsn Arg Arg Pro 305 310 315 320 Ile Leu Ile Ile Val Thr Leu Glu Thr ArgAsp Gly Gln Val Leu Gly 325 330 335 Arg Arg Cys Phe Glu Ala Arg Ile CysAla Cys Pro Gly Arg Asp Arg 340 345 350 Lys Ala Asp Glu Asp Ser Ile ArgLys Gln Gln Val Ser Asp Ser Ala 355 360 365 Lys Asn Gly Asp Gly Thr LysArg Pro Phe Arg Gln Asn Thr His Gly 370 375 380 Ile Gln Met Thr Ser IleLys Lys Arg Arg Ser Pro Asp Asp Glu Leu 385 390 395 400 Leu Tyr Leu ProVal Arg Gly Arg Glu Thr Tyr Glu Met Leu Leu Lys 405 410 415 Ile Lys GluSer Leu Glu Leu Met Gln Tyr Leu Pro Gln His Thr Ile 420 425 430 Glu ThrTyr Arg Gln Gln Gln Gln Gln Gln His Gln His Leu Leu Gln 435 440 445 LysGln Thr Ser Met Gln Ser Gln Ser Ser Tyr Gly Asn Ser Ser Pro 450 455 460Pro Leu Asn Lys Met Asn Ser Met Asn Lys Leu Pro Ser Val Ser Gln 465 470475 480 Leu Ile Asn Pro Gln Gln Arg Asn Ala Leu Thr Pro Thr Thr Met Pro485 490 495 Glu Gly Met Gly Ala Asn Ile Pro Met Met Gly Thr His Met ProMet 500 505 510 Ala Gly Asp Met Asn Gly Leu Ser Pro Thr Gln Ala Leu ProPro Pro 515 520 525 Leu Ser Met Pro Ser Thr Ser His Cys Thr Pro Pro ProPro Tyr Pro 530 535 540 Thr Asp Cys Ser Ile Val Ser Phe Leu Ala Arg LeuGly Cys Ser Ser 545 550 555 560 Cys Leu Asp Tyr Phe Thr Thr Gln Gly LeuThr Thr Ile Tyr Gln Ile 565 570 575 Glu His Tyr Ser Met Asp Asp Leu AlaSer Leu Lys Ile Pro Glu Gln 580 585 590 Phe Arg His Ala Ile Trp Lys GlyIle Leu Asp His Arg Gln Leu His 595 600 605 Asp Phe Ser Ser Pro Pro HisLeu Leu Arg Thr Pro Ser Gly Ala Ser 610 615 620 Thr Val Ser Val Gly SerSer Glu Thr Arg Gly Glu Arg Val Ile Asp 625 630 635 640 Ala Val Arg PheThr Leu Arg Gln Thr Ile Ser Phe Pro Pro Arg Asp 645 650 655 Glu Trp AsnAsp Phe Asn Phe Asp Met Asp Ser Arg Arg Asn Lys Gln 660 665 670 Gln ArgIle Lys Glu Glu Gly Glu 675 680 20 555 PRT murine 20 Met Asn Phe Glu ThrSer Arg Cys Ala Thr Leu Gln Tyr Cys Pro Asp 1 5 10 15 Pro Tyr Ile GlnArg Phe Ile Glu Thr Pro Ala His Phe Ser Trp Lys 20 25 30 Glu Ser Tyr TyrArg Ser Ala Met Ser Gln Ser Thr Gln Thr Ser Glu 35 40 45 Phe Leu Ser ProGlu Val Phe Gln His Ile Trp Asp Phe Leu Glu Gln 50 55 60 Pro Ile Cys SerVal Gln Pro Ile Glu Leu Asn Phe Val Asp Glu Pro 65 70 75 80 Ser Glu AsnGly Ala Thr Asn Lys Ile Glu Ile Ser Met Asp Cys Ile 85 90 95 Arg Met GlnAsp Ser Asp Leu Ser Asp Pro Met Trp Pro Gln Tyr Thr 100 105 110 Asn LeuGly Leu Leu Asn Ser Met Asp Gln Gln Ile Gln Asn Gly Ser 115 120 125 SerSer Thr Ser Pro Tyr Asn Thr Asp His Ala Gln Asn Ser Val Thr 130 135 140Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe Asp Ala Leu Ser 145 150155 160 Pro Ser Pro Ala Ile Pro Ser Asn Thr Asp Tyr Pro Gly Pro His Ser165 170 175 Phe Asp Val Ser Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala ThrTrp 180 185 190 Thr Tyr Ser Thr Glu Leu Lys Lys Leu Tyr Cys Gln Ile AlaLys Thr 195 200 205 Cys Pro Ile Gln Ile Lys Val Met Thr Pro Pro Pro GlnGly Ala Val 210 215 220 Ile Arg Ala Met Pro Val Tyr Lys Lys Ala Glu HisVal Thr Glu Val 225 230 235 240 Val Lys Arg Cys Pro Asn His Glu Leu SerArg Glu Phe Asn Glu Gly 245 250 255 Gln Ile Ala Pro Pro Ser His Leu IleArg Val Glu Gly Asn Ser His 260 265 270 Ala Gln Tyr Val Glu Asp Pro IleThr Gly Arg Gln Ser Val Leu Val 275 280 285 Pro Tyr Glu Pro Pro Gln ValGly Thr Glu Phe Thr Thr Val Leu Tyr 290 295 300 Asn Phe Met Cys Asn SerSer Cys Val Gly Gly Met Asn Arg Arg Pro 305 310 315 320 Ile Leu Ile IleVal Thr Leu Glu Thr Arg Asp Gly Gln Val Leu Gly 325 330 335 Arg Arg CysPhe Glu Ala Arg Ile Cys Ala Cys Pro Gly Arg Asp Arg 340 345 350 Lys AlaAsp Glu Asp Ser Ile Arg Lys Gln Gln Val Ser Asp Ser Ala 355 360 365 LysAsn Gly Asp Gly Thr Lys Arg Pro Phe Arg Gln Asn Thr His Gly 370 375 380Ile Gln Met Thr Ser Ile Lys Lys Arg Arg Ser Pro Asp Asp Glu Leu 385 390395 400 Leu Tyr Leu Pro Val Arg Gly Arg Glu Thr Tyr Glu Met Leu Leu Lys405 410 415 Ile Lys Glu Ser Leu Glu Leu Met Gln Tyr Leu Pro Gln His ThrIle 420 425 430 Glu Thr Tyr Arg Gln Gln Gln Gln Gln Gln His Gln His LeuLeu Gln 435 440 445 Lys Gln Thr Ser Met Gln Ser Gln Ser Ser Tyr Gly AsnSer Ser Pro 450 455 460 Pro Leu Asn Lys Met Asn Ser Met Asn Lys Leu ProSer Val Ser Gln 465 470 475 480 Leu Ile Asn Pro Gln Gln Arg Asn Ala LeuThr Pro Thr Thr Met Pro 485 490 495 Glu Gly Met Gly Ala Asn Ile Pro MetMet Gly Thr His Met Pro Met 500 505 510 Ala Gly Asp Met Asn Gly Leu SerPro Thr Gln Ala Leu Pro Pro Pro 515 520 525 Leu Ser Met Pro Ser Thr SerHis Cys Thr Pro Pro Pro Pro Tyr Pro 530 535 540 Thr Asp Cys Ser Ile ValArg Ile Trp Gln Val 545 550 555 21 483 PRT murine 21 Met Asn Phe Glu ThrSer Arg Cys Ala Thr Leu Gln Tyr Cys Pro Asp 1 5 10 15 Pro Tyr Ile GlnArg Phe Ile Glu Thr Pro Ala His Phe Ser Trp Lys 20 25 30 Glu Ser Tyr TyrArg Ser Ala Met Ser Gln Ser Thr Gln Thr Ser Glu 35 40 45 Phe Leu Ser ProGlu Val Phe Gln His Ile Trp Asp Phe Leu Glu Gln 50 55 60 Pro Ile Cys SerVal Gln Pro Ile Glu Leu Asn Phe Val Asp Glu Pro 65 70 75 80 Ser Glu AsnGly Ala Thr Asn Lys Ile Glu Ile Ser Met Asp Cys Ile 85 90 95 Arg Met GlnAsp Ser Asp Leu Ser Asp Pro Met Trp Pro Gln Tyr Thr 100 105 110 Asn LeuGly Leu Leu Asn Ser Met Asp Gln Gln Ile Gln Asn Gly Ser 115 120 125 SerSer Thr Ser Pro Tyr Asn Thr Asp His Ala Gln Asn Ser Val Thr 130 135 140Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe Asp Ala Leu Ser 145 150155 160 Pro Ser Pro Ala Ile Pro Ser Asn Thr Asp Tyr Pro Gly Pro His Ser165 170 175 Phe Asp Val Ser Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala ThrTrp 180 185 190 Thr Tyr Ser Thr Glu Leu Lys Lys Leu Tyr Cys Gln Ile AlaLys Thr 195 200 205 Cys Pro Ile Gln Ile Lys Val Met Thr Pro Pro Pro GlnGly Ala Val 210 215 220 Ile Arg Ala Met Pro Val Tyr Lys Lys Ala Glu HisVal Thr Glu Val 225 230 235 240 Val Lys Arg Cys Pro Asn His Glu Leu SerArg Glu Phe Asn Glu Gly 245 250 255 Gln Ile Ala Pro Pro Ser His Leu IleArg Val Glu Gly Asn Ser His 260 265 270 Ala Gln Tyr Val Glu Asp Pro IleThr Gly Arg Gln Ser Val Leu Val 275 280 285 Pro Tyr Glu Pro Pro Gln ValGly Thr Glu Phe Thr Thr Val Leu Tyr 290 295 300 Asn Phe Met Cys Asn SerSer Cys Val Gly Gly Met Asn Arg Arg Pro 305 310 315 320 Ile Leu Ile IleVal Thr Leu Glu Thr Arg Asp Gly Gln Val Leu Gly 325 330 335 Arg Arg CysPhe Glu Ala Arg Ile Cys Ala Cys Pro Gly Arg Asp Arg 340 345 350 Lys AlaAsp Glu Asp Ser Ile Arg Lys Gln Gln Val Ser Asp Ser Ala 355 360 365 LysAsn Gly Asp Ala Phe Arg Gln Asn Thr His Gly Ile Gln Met Thr 370 375 380Ser Ile Lys Lys Arg Arg Ser Pro Asp Asp Glu Leu Leu Tyr Leu Pro 385 390395 400 Val Arg Gly Arg Glu Thr Tyr Glu Met Leu Leu Lys Ile Lys Glu Ser405 410 415 Leu Glu Leu Met Gln Tyr Leu Pro Gln His Thr Ile Glu Thr TyrArg 420 425 430 Gln Gln Gln Gln Gln Gln His Gln His Leu Leu Gln Lys HisLeu Leu 435 440 445 Ser Ala Cys Phe Arg Asn Glu Leu Val Glu Pro Arg GlyGlu Ala Pro 450 455 460 Thr Gln Ser Asp Val Phe Phe Arg His Ser Asn ProPro Asn His Ser 465 470 475 480 Val Tyr Pro 22 586 PRT murine 22 Met LeuTyr Leu Glu Asn Asn Ala Gln Thr Gln Phe Ser Glu Pro Gln 1 5 10 15 TyrThr Asn Leu Gly Leu Leu Asn Ser Met Asp Gln Gln Ile Gln Asn 20 25 30 GlySer Ser Ser Thr Ser Pro Tyr Asn Thr Asp His Ala Gln Asn Ser 35 40 45 ValThr Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe Asp Ala 50 55 60 LeuSer Pro Ser Pro Ala Ile Pro Ser Asn Thr Asp Tyr Pro Gly Pro 65 70 75 80His Ser Phe Asp Val Ser Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala 85 90 95Thr Trp Thr Tyr Ser Thr Glu Leu Lys Lys Leu Tyr Cys Gln Ile Ala 100 105110 Lys Thr Cys Pro Ile Gln Ile Lys Val Met Thr Pro Pro Pro Gln Gly 115120 125 Ala Val Ile Arg Ala Met Pro Val Tyr Lys Lys Ala Glu His Val Thr130 135 140 Glu Val Val Lys Arg Cys Pro Asn His Glu Leu Ser Arg Glu PheAsn 145 150 155 160 Glu Gly Gln Ile Ala Pro Pro Ser His Leu Ile Arg ValGlu Gly Asn 165 170 175 Ser His Ala Gln Tyr Val Glu Asp Pro Ile Thr GlyArg Gln Ser Val 180 185 190 Leu Val Pro Tyr Glu Pro Pro Gln Val Gly ThrGlu Phe Thr Thr Val 195 200 205 Leu Tyr Asn Phe Met Cys Asn Ser Ser CysVal Gly Gly Met Asn Arg 210 215 220 Arg Pro Ile Leu Ile Ile Val Thr LeuGlu Thr Arg Asp Gly Gln Val 225 230 235 240 Leu Gly Arg Arg Cys Phe GluAla Arg Ile Cys Ala Cys Pro Gly Arg 245 250 255 Asp Arg Lys Ala Asp GluAsp Ser Ile Arg Lys Gln Gln Val Ser Asp 260 265 270 Ser Ala Lys Asn GlyAsp Gly Thr Lys Arg Pro Phe Arg Gln Asn Thr 275 280 285 His Gly Ile GlnMet Thr Ser Ile Lys Lys Arg Arg Ser Pro Asp Asp 290 295 300 Glu Leu LeuTyr Leu Pro Val Arg Gly Arg Glu Thr Tyr Glu Met Leu 305 310 315 320 LeuLys Ile Lys Glu Ser Leu Glu Leu Met Gln Tyr Leu Pro Gln His 325 330 335Thr Ile Glu Thr Tyr Arg Gln Gln Gln Gln Gln Gln His Gln His Leu 340 345350 Leu Gln Lys Gln Thr Ser Met Gln Ser Gln Ser Ser Tyr Gly Asn Ser 355360 365 Ser Pro Pro Leu Asn Lys Met Asn Ser Met Asn Lys Leu Pro Ser Val370 375 380 Ser Gln Leu Ile Asn Pro Gln Gln Arg Asn Ala Leu Thr Pro ThrThr 385 390 395 400 Met Pro Glu Gly Met Gly Ala Asn Ile Pro Met Met GlyThr His Met 405 410 415 Pro Met Ala Gly Asp Met Asn Gly Leu Ser Pro ThrGln Ala Leu Pro 420 425 430 Pro Pro Leu Ser Met Pro Ser Thr Ser His CysThr Pro Pro Pro Pro 435 440 445 Tyr Pro Thr Asp Cys Ser Ile Val Ser PheLeu Ala Arg Leu Gly Cys 450 455 460 Ser Ser Cys Leu Asp Tyr Phe Thr ThrGln Gly Leu Thr Thr Ile Tyr 465 470 475 480 Gln Ile Glu His Tyr Ser MetAsp Asp Leu Ala Ser Leu Lys Ile Pro 485 490 495 Glu Gln Phe Arg His AlaIle Trp Lys Gly Ile Leu Asp His Arg Gln 500 505 510 Leu His Asp Phe SerSer Pro Pro His Leu Leu Arg Thr Pro Ser Gly 515 520 525 Ala Ser Thr ValSer Val Gly Ser Ser Glu Thr Arg Gly Glu Arg Val 530 535 540 Ile Asp AlaVal Arg Phe Thr Leu Arg Gln Thr Ile Ser Phe Pro Pro 545 550 555 560 ArgAsp Glu Trp Asn Asp Phe Asn Phe Asp Met Asp Ser Arg Arg Asn 565 570 575Lys Gln Gln Arg Ile Lys Glu Glu Gly Glu 580 585 23 461 PRT murine 23 MetLeu Tyr Leu Glu Asn Asn Ala Gln Thr Gln Phe Ser Glu Pro Gln 1 5 10 15Tyr Thr Asn Leu Gly Leu Leu Asn Ser Met Asp Gln Gln Ile Gln Asn 20 25 30Gly Ser Ser Ser Thr Ser Pro Tyr Asn Thr Asp His Ala Gln Asn Ser 35 40 45Val Thr Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe Asp Ala 50 55 60Leu Ser Pro Ser Pro Ala Ile Pro Ser Asn Thr Asp Tyr Pro Gly Pro 65 70 7580 His Ser Phe Asp Val Ser Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala 85 9095 Thr Trp Thr Tyr Ser Thr Glu Leu Lys Lys Leu Tyr Cys Gln Ile Ala 100105 110 Lys Thr Cys Pro Ile Gln Ile Lys Val Met Thr Pro Pro Pro Gln Gly115 120 125 Ala Val Ile Arg Ala Met Pro Val Tyr Lys Lys Ala Glu His ValThr 130 135 140 Glu Val Val Lys Arg Cys Pro Asn His Glu Leu Ser Arg GluPhe Asn 145 150 155 160 Glu Gly Gln Ile Ala Pro Pro Ser His Leu Ile ArgVal Glu Gly Asn 165 170 175 Ser His Ala Gln Tyr Val Glu Asp Pro Ile ThrGly Arg Gln Ser Val 180 185 190 Leu Val Pro Tyr Glu Pro Pro Gln Val GlyThr Glu Phe Thr Thr Val 195 200 205 Leu Tyr Asn Phe Met Cys Asn Ser SerCys Val Gly Gly Met Asn Arg 210 215 220 Arg Pro Ile Leu Ile Ile Val ThrLeu Glu Thr Arg Asp Gly Gln Val 225 230 235 240 Leu Gly Arg Arg Cys PheGlu Ala Arg Ile Cys Ala Cys Pro Gly Arg 245 250 255 Asp Arg Lys Ala AspGlu Asp Ser Ile Arg Lys Gln Gln Val Ser Asp 260 265 270 Ser Ala Lys AsnGly Asp Gly Thr Lys Arg Pro Phe Arg Gln Asn Thr 275 280 285 His Gly IleGln Met Thr Ser Ile Lys Lys Arg Arg Ser Pro Asp Asp 290 295 300 Glu LeuLeu Tyr Leu Pro Val Arg Gly Arg Glu Thr Tyr Glu Met Leu 305 310 315 320Leu Lys Ile Lys Glu Ser Leu Glu Leu Met Gln Tyr Leu Pro Gln His 325 330335 Thr Ile Glu Thr Tyr Arg Gln Gln Gln Gln Gln Gln His Gln His Leu 340345 350 Leu Gln Lys Gln Thr Ser Met Gln Ser Gln Ser Ser Tyr Gly Asn Ser355 360 365 Ser Pro Pro Leu Asn Lys Met Asn Ser Met Asn Lys Leu Pro SerVal 370 375 380 Ser Gln Leu Ile Asn Pro Gln Gln Arg Asn Ala Leu Thr ProThr Thr 385 390 395 400 Met Pro Glu Gly Met Gly Ala Asn Ile Pro Met MetGly Thr His Met 405 410 415 Pro Met Ala Gly Asp Met Asn Gly Leu Ser ProThr Gln Ala Leu Pro 420 425 430 Pro Pro Leu Ser Met Pro Ser Thr Ser HisCys Thr Pro Pro Pro Pro 435 440 445 Tyr Pro Thr Asp Cys Ser Ile Val ArgIle Trp Gln Val 450 455 460 24 389 PRT murine 24 Met Leu Tyr Leu Glu AsnAsn Ala Gln Thr Gln Phe Ser Glu Pro Gln 1 5 10 15 Tyr Thr Asn Leu GlyLeu Leu Asn Ser Met Asp Gln Gln Ile Gln Asn 20 25 30 Gly Ser Ser Ser ThrSer Pro Tyr Asn Thr Asp His Ala Gln Asn Ser 35 40 45 Val Thr Ala Pro SerPro Tyr Ala Gln Pro Ser Ser Thr Phe Asp Ala 50 55 60 Leu Ser Pro Ser ProAla Ile Pro Ser Asn Thr Asp Tyr Pro Gly Pro 65 70 75 80 His Ser Phe AspVal Ser Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala 85 90 95 Thr Trp Thr TyrSer Thr Glu Leu Lys Lys Leu Tyr Cys Gln Ile Ala 100 105 110 Lys Thr CysPro Ile Gln Ile Lys Val Met Thr Pro Pro Pro Gln Gly 115 120 125 Ala ValIle Arg Ala Met Pro Val Tyr Lys Lys Ala Glu His Val Thr 130 135 140 GluVal Val Lys Arg Cys Pro Asn His Glu Leu Ser Arg Glu Phe Asn 145 150 155160 Glu Gly Gln Ile Ala Pro Pro Ser His Leu Ile Arg Val Glu Gly Asn 165170 175 Ser His Ala Gln Tyr Val Glu Asp Pro Ile Thr Gly Arg Gln Ser Val180 185 190 Leu Val Pro Tyr Glu Pro Pro Gln Val Gly Thr Glu Phe Thr ThrVal 195 200 205 Leu Tyr Asn Phe Met Cys Asn Ser Ser Cys Val Gly Gly MetAsn Arg 210 215 220 Arg Pro Ile Leu Ile Ile Val Thr Leu Glu Thr Arg AspGly Gln Val 225 230 235 240 Leu Gly Arg Arg Cys Phe Glu Ala Arg Ile CysAla Cys Pro Gly Arg 245 250 255 Asp Arg Lys Ala Asp Glu Asp Ser Ile ArgLys Gln Gln Val Ser Asp 260 265 270 Ser Ala Lys Asn Gly Asp Ala Phe ArgGln Asn Thr His Gly Ile Gln 275 280 285 Met Thr Ser Ile Lys Lys Arg ArgSer Pro Asp Asp Glu Leu Leu Tyr 290 295 300 Leu Pro Val Arg Gly Arg GluThr Tyr Glu Met Leu Leu Lys Ile Lys 305 310 315 320 Glu Ser Leu Glu LeuMet Gln Tyr Leu Pro Gln His Thr Ile Glu Thr 325 330 335 Tyr Arg Gln GlnGln Gln Gln Gln His Gln His Leu Leu Gln Lys His 340 345 350 Leu Leu SerAla Cys Phe Arg Asn Glu Leu Val Glu Pro Arg Gly Glu 355 360 365 Ala ProThr Gln Ser Asp Val Phe Phe Arg His Ser Asn Pro Pro Asn 370 375 380 HisSer Val Tyr Pro 385 25 393 PRT Homo sapiens 25 Met Glu Glu Pro Gln SerAsp Pro Ser Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser AspLeu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro Leu Pro SerGln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45 Asp Ile Glu Gln TrpPhe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60 Arg Met Pro Glu AlaAla Pro Pro Val Ala Pro Ala Pro Ala Ala Pro 65 70 75 80 Thr Pro Ala AlaPro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser 85 90 95 Val Pro Ser GlnLys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110 Phe Leu HisSer Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125 Ala LeuAsn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln 130 135 140 LeuTrp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155160 Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys 165170 175 Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln180 185 190 His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu AspAsp 195 200 205 Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu ProPro Glu 210 215 220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr MetCys Asn Ser 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro IleLeu Thr Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu Leu GlyArg Asn Ser Phe Glu Val 260 265 270 His Val Cys Ala Cys Pro Gly Arg AspArg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys Lys Gly Glu Pro HisHis Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys Arg Ala Leu Pro Asn AsnThr Ser Ser Ser Pro Gln Pro Lys Lys 305 310 315 320 Lys Pro Leu Asp GlyGlu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu 325 330 335 Arg Phe Glu MetPhe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 340 345 350 Ala Gln AlaGly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365 Leu LysSer Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met 370 375 380 PheLys Thr Glu Gly Pro Asp Ser Asp 385 390 26 499 PRT Homo sapiens 26 MetAla Gln Ser Thr Ala Thr Ser Pro Asp Gly Gly Thr Thr Phe Glu 1 5 10 15His Leu Trp Ser Ser Leu Glu Pro Asp Ser Thr Tyr Phe Asp Leu Pro 20 25 30Gln Ser Ser Arg Gly Asn Asn Glu Val Val Gly Gly Thr Asp Ser Ser 35 40 45Met Asp Val Phe His Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln 50 55 60Phe Asn Leu Leu Ser Ser Thr Met Asp Gln Met Ser Ser Arg Ala Ala 65 70 7580 Ser Ala Ser Pro Tyr Thr Pro Glu His Ala Ala Ser Val Pro Thr His 85 9095 Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe Asp Thr Met Ser Pro Ala 100105 110 Pro Val Ile Pro Ser Asn Thr Asp Tyr Pro Gly Pro His His Phe Glu115 120 125 Val Thr Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala Thr Trp ThrTyr 130 135 140 Ser Pro Leu Leu Lys Lys Leu Tyr Cys Gln Ile Ala Lys ThrCys Pro 145 150 155 160 Ile Gln Ile Lys Val Ser Thr Pro Pro Pro Pro GlyThr Ala Ile Arg 165 170 175 Ala Met Pro Val Tyr Lys Lys Ala Glu His ValThr Asp Val Val Lys 180 185 190 Arg Cys Pro Asn His Glu Leu Gly Arg AspPhe Asn Glu Gly Gln Ser 195 200 205 Ala Pro Ala Ser His Leu Ile Arg ValGlu Gly Asn Asn Leu Ser Gln 210 215 220 Tyr Val Asp Asp Pro Val Thr GlyArg Gln Ser Val Val Val Pro Tyr 225 230 235 240 Glu Pro Pro Gln Val GlyThr Glu Phe Thr Thr Ile Leu Tyr Asn Phe 245 250 255 Met Cys Asn Ser SerCys Val Gly Gly Met Asn Arg Arg Pro Ile Leu 260 265 270 Ile Ile Ile ThrLeu Glu Met Arg Asp Gly Gln Val Leu Gly Arg Arg 275 280 285 Ser Phe GluGly Arg Ile Cys Ala Cys Pro Gly Arg Asp Arg Lys Ala 290 295 300 Asp GluAsp His Tyr Arg Glu Gln Gln Ala Leu Asn Glu Ser Ser Ala 305 310 315 320Lys Asn Gly Ala Ala Ser Lys Arg Ala Phe Lys Gln Ser Pro Pro Ala 325 330335 Val Pro Ala Leu Gly Ala Gly Val Lys Lys Arg Arg His Gly Asp Glu 340345 350 Asp Thr Tyr Tyr Leu Gln Val Arg Gly Arg Glu Asn Phe Glu Ile Leu355 360 365 Met Lys Leu Lys Glu Ser Leu Glu Leu Met Glu Leu Val Pro GlnPro 370 375 380 Leu Val Asp Ser Tyr Arg Gln Gln Gln Gln Leu Leu Gln ArgPro Ser 385 390 395 400 His Leu Gln Pro Pro Ser Tyr Gly Pro Val Leu SerPro Met Asn Lys 405 410 415 Val His Gly Gly Met Asn Lys Leu Pro Ser ValAsn Gln Leu Val Gly 420 425 430 Gln Pro Pro Pro His Ser Ser Ala Ala ThrPro Asn Leu Gly Pro Val 435 440 445 Gly Pro Gly Met Leu Asn Asn His GlyHis Ala Val Pro Ala Asn Gly 450 455 460 Glu Met Ser Ser Ser His Ser AlaGln Ser Met Val Ser Gly Ser His 465 470 475 480 Cys Thr Pro Pro Pro ProTyr His Ala Asp Pro Ser Leu Val Arg Thr 485 490 495 Trp Gly Pro 27 29DNA Artificial Sequence Description of Artificial Sequence syntheticprimer 27 ggcctcgagt acaantwcat gtgtaayag 29 28 29 DNA ArtificialSequence Description of Artificial Sequence synthetic primer 28ggcatcgatt ctcttccagg gcaagcaca 29 29 27 DNA Artificial SequenceDescription of Artificial Sequence synthetic primer 29 ggcatcgatgaactcacggc tcagctc 27 30 43 DNA Artificial Sequence Description ofArtificial Sequence synthetic primer 30 tttagtgagg gttaataagc ggccgcgtcgtgactgggag cgc 43 31 31 DNA Artificial Sequence Description ofArtificial Sequence synthetic primer 31 gccctggagg cggccgctta ttaaccctcac 31 32 27 DNA Artificial Sequence Description of Artificial Sequencesynthetic primer 32 ggcatcgatg tagacaggca tggcacg 27 33 27 DNAArtificial Sequence Description of Artificial Sequence synthetic primer33 gggctcgagc tgaagaagct gtactgc 27 34 27 DNA Artificial SequenceDescription of Artificial Sequence synthetic primer 34 gggatcgatctccgtttctt gatggaa 27 35 19 DNA Artificial Sequence Description ofArtificial Sequence synthetic primer 35 cctgcctgga cttgcctgg 19 36 19DNA Artificial Sequence Description of Artificial Sequence syntheticprimer 36 ccaggcaagt ccaggcagg 19 37 20 DNA Artificial SequenceDescription of Artificial Sequence synthetic primer 37 gaacatgtcccaacatgttg 20 38 20 DNA Artificial Sequence Description of ArtificialSequence synthetic primer 38 caacatgttg ggacatgttc 20 39 19 DNAArtificial Sequence Description of Artificial Sequence synthetic primer39 ccttaatgga ctttaatgg 19 40 19 DNA Artificial Sequence Description ofArtificial Sequence synthetic primer 40 ccattaaagt ccattaagg 19 41 19DNA Artificial Sequence Description of Artificial Sequence syntheticprimer 41 atgtcccaga gccacacag 19 42 18 DNA Artificial SequenceDescription of Artificial Sequence synthetic primer 42 agctcatggttggggcac 18 43 18 DNA Artificial Sequence Description of ArtificialSequence synthetic primer 43 cagactcaat ttagtgag 18 44 18 DNA ArtificialSequence Description of Artificial Sequence synthetic primer 44agctcatggt tggggcac 18 45 120 PRT murine 45 Met Asn Phe Glu Thr Ser ArgCys Ala Thr Leu Gln Tyr Cys Pro Asp 1 5 10 15 Pro Tyr Ile Gln Arg PheIle Glu Thr Pro Ala His Phe Ser Trp Lys 20 25 30 Glu Ser Tyr Tyr Arg SerAla Met Ser Gln Ser Thr Gln Thr Ser Glu 35 40 45 Phe Leu Ser Pro Glu ValPhe Gln His Ile Trp Asp Phe Leu Glu Gln 50 55 60 Pro Ile Cys Ser Val GlnPro Ile Glu Leu Asn Phe Val Asp Glu Pro 65 70 75 80 Ser Glu Asn Gly AlaThr Asn Lys Ile Glu Ile Ser Met Asp Cys Ile 85 90 95 Arg Met Gln Asp SerAsp Leu Ser Asp Pro Met Trp Pro Gln Tyr Thr 100 105 110 Asn Leu Gly LeuLeu Asn Ser Met 115 120 46 81 PRT Homo sapiens 46 Met Ser Gln Ser ThrGln Thr Asn Glu Phe Leu Ser Pro Glu Val Phe 1 5 10 15 Gln His Ile TrpAsp Phe Leu Glu Gln Pro Ile Cys Ser Val Gln Pro 20 25 30 Ile Asp Leu AsnPhe Val Asp Glu Pro Ser Glu Asp Gly Ala Thr Asn 35 40 45 Lys Ile Glu IleSer Met Asp Cys Ile Arg Met Gln Asp Ser Asp Leu 50 55 60 Ser Asp Pro MetTrp Pro Gln Tyr Thr Asn Leu Gly Leu Leu Asn Ser 65 70 75 80 Met 47 26PRT Homo sapiens 47 Met Leu Tyr Leu Glu Asn Asn Ala Gln Thr Gln Phe SerGlu Pro Gln 1 5 10 15 Tyr Thr Asn Leu Gly Leu Leu Asn Ser Met 20 25 48245 PRT Homo sapiens 48 Arg Gln Gln Gln Gln Gln Gln His Gln His Leu LeuGln Lys Gln Thr 1 5 10 15 Ser Ile Gln Ser Pro Ser Ser Tyr Gly Asn SerSer Pro Pro Leu Asn 20 25 30 Lys Met Asn Ser Met Asn Lys Leu Pro Ser ValSer Gln Leu Ile Asn 35 40 45 Pro Gln Gln Arg Asn Ala Leu Thr Pro Thr ThrIle Pro Asp Gly Met 50 55 60 Gly Ala Asn Ile Pro Met Met Gly Thr His MetPro Met Ala Gly Asp 65 70 75 80 Met Asn Gly Leu Ser Pro Thr Gln Ala LeuPro Pro Pro Leu Ser Met 85 90 95 Pro Ser Thr Ser His Cys Thr Pro Pro ProPro Tyr Pro Thr Asp Cys 100 105 110 Ser Ile Val Ser Phe Leu Ala Arg LeuGly Cys Ser Ser Cys Leu Asp 115 120 125 Tyr Phe Thr Thr Gln Gly Leu ThrThr Ile Tyr Gln Ile Glu His Tyr 130 135 140 Ser Met Asp Asp Leu Ala SerLeu Lys Ile Pro Glu Gln Phe Arg His 145 150 155 160 Ala Ile Trp Lys GlyIle Leu Asp His Arg Gln Leu His Glu Phe Ser 165 170 175 Ser Pro Ser HisLeu Leu Arg Thr Pro Ser Ser Ala Ser Thr Val Ser 180 185 190 Val Gly SerSer Glu Thr Arg Gly Glu Arg Val Ile Asp Ala Val Arg 195 200 205 Phe ThrLeu Arg Gln Thr Ile Ser Phe Pro Pro Arg Asp Glu Trp Asn 210 215 220 AspPhe Asn Phe Asp Met Asp Ala Arg Arg Asn Lys Gln Gln Arg Ile 225 230 235240 Lys Glu Glu Gly Glu 245 49 120 PRT Homo sapiens 49 Arg Gln Gln GlnGln Gln Gln His Gln His Leu Leu Gln Lys Gln Thr 1 5 10 15 Ser Ile GlnSer Pro Ser Ser Tyr Gly Asn Ser Ser Pro Pro Leu Asn 20 25 30 Lys Met AsnSer Met Asn Lys Leu Pro Ser Val Ser Gln Leu Ile Asn 35 40 45 Pro Gln GlnArg Asn Ala Leu Thr Pro Thr Thr Ile Pro Asp Gly Met 50 55 60 Gly Ala AsnIle Pro Met Met Gly Thr His Met Pro Met Ala Gly Asp 65 70 75 80 Met AsnGly Leu Ser Pro Thr Gln Ala Leu Pro Pro Pro Leu Ser Met 85 90 95 Pro SerThr Ser His Cys Thr Pro Pro Pro Pro Tyr Pro Thr Asp Cys 100 105 110 SerIle Val Arg Ile Trp Gln Val 115 120 50 52 PRT Homo sapiens 50 Arg GlnGln Gln Gln Gln Gln His Gln His Leu Leu Gln Lys His Leu 1 5 10 15 LeuSer Ala Cys Phe Arg Asn Glu Leu Val Glu Pro Arg Arg Glu Thr 20 25 30 ProLys Gln Ser Asp Val Phe Phe Arg His Ser Lys Pro Pro Asn Arg 35 40 45 SerVal Tyr Pro 50

We claim:
 1. An isolated antibody that binds to a p63 protein and doesnot detectably bind to a p53 or p73 protein.
 2. The antibody of claim 1,wherein the antibody is specifically immunoreactive with a p63 protein.3. The antibody of claim 1, wherein the antibody binds to a p63 proteincomprising an amino acid sequence having at least about 90% identitywith a sequence set forth in any one of SEQ ID NOs: 13-24.
 4. Theantibody of claim 3, wherein the antibody binds to a p63 proteincomprising an amino acid sequence having at least about 95% identitywith a sequence set forth in any one of SEQ ID NOs: 13-24.
 5. Theantibody of claim 4, wherein the antibody binds to a p63 proteincomprising an amino acid sequence having at least about 98% identitywith a sequence set forth in any one of SEQ ID NOs: 13-24.
 6. Theantibody of claim 3, wherein the antibody binds to a p63 proteincomprising an amino acid sequence having a sequence set forth in any oneof SEQ ID NOs: 13-24.
 7. The antibody of claim 1, wherein the antibodybinds to a p63 protein encoded by a nucleic acid sequence whichhybridizes under stringent conditions to the complementary strand of anucleic acid having a sequence set forth in any one of SEQ ID NOs: 1-12.8. The antibody of claim 7, wherein the antibody binds to a p63 proteinencoded by a nucleic acid sequence of any one of SEQ ID NOs: 1-12. 9.The antibody of claim 1, wherein the antibody binds to a p63 proteincomprising the amino acid sequence set forth in SEQ ID NO:
 16. 10. Theantibody of claim 1, wherein binding is determined usingimmunoprecipitation, immunostaining, western blotting, or aradioimmunoassay (RIA).
 11. The antibody of claim 1, wherein theantibody is a monoclonal antibody.
 12. The antibody of claim 1, whereinthe antibody is a recombinant antibody.
 13. The antibody of claim 1,wherein the antibody is an antibody fragment.
 14. The antibody of claim13, wherein the antibody fragment is an Fab, F(ab′)₂, Fab′, Fv, or scFv.15. The antibody of claim 1, wherein the antibody is a mouse antibody.16. The antibody of claim 1, wherein the antibody is a human antibody.17. The antibody of claim 1, wherein the antibody is a humanizedantibody.
 18. The antibody of claim 1, wherein the antibody comprises alabel.
 19. The antibody of claim 18, wherein the label is at least oneof the following: a radioisotope, a fluorescent compound, an enzyme, oran enzyme co-factor.
 20. The antibody of claim 1, wherein the antibodyis antibody 4A4.
 21. The antibody of claim 1, wherein the antibody bindsto the epitope to which antibody 4A4 binds.
 22. The antibody of claim 1,wherein the antibody inhibits binding of p63 to antibody 4A4.
 23. Anisolated antibody that selectively binds to a p63 protein relative top53 and p73 proteins.
 24. A hybridoma producing the antibody of claim11.
 25. A purified preparation of polyclonal antibodies wherein theantibodies bind to a p63 protein and do not detectably bind to a p53 orp73 protein.
 26. The purified preparation of claim 25, wherein theantibodies are specifically immunoreactive with a p63 protein.
 27. Thepurified preparation of claim 25, wherein the antibodies are antibodyfragments.
 28. The antibody of claim 25, wherein binding is determinedusing immunoprecipitation, immunostaining, western blotting, or aradioimmunoassay (RIA).
 29. An isolated polypeptide wherein saidpolypeptide comprises (a) an amino acid sequence set forth in any one ofSEQ ID NOs: 13-24; (b) an amino acid sequence having at least about 90%identity with an amino acid sequence set forth in any one of SEQ ID NOs:13-24; (c) an amino acid sequence encoded by a nucleic acid thathybridizes under stringent conditions to the complementary strand of anucleic acid having a sequence set forth in any one of SEQ ID NOs: 1-12;or (d) a fragment of an amino acid sequence set forth in any one of SEQID NOs: 13-24.
 30. The isolated polypeptide of claim 29, wherein saidpolypeptide has one or more of the following biological activities: (i)binds a target DNA sequence, (ii) transactivates a target gene, (iii)induces apoptosis, (iv) oligomerizes, (v) localizes to basal epithelialcells, or (vi) localizes to squamous cervical cells.
 31. The isolatedpolypeptide of claim 30, wherein said target DNA sequence is at leastone of the following: (i) a p53-responsive element, (ii) a minimal p53target binding sequence, or (iii) a p53 target DNA binding sequence in ap21 promoter.
 32. The isolated polypeptide of claim 29, wherein saidpolypeptide is a fusion protein.
 33. The isolated polypeptide of claim32, wherein said polypeptide is functional in a two-hybrid assay. 34.The isolated polypeptide of claim 29, wherein said polypeptide isfunctions either as an agonist of cell cycle regulation or an antagonistof cell cycle regulation.
 35. The isolated polypeptide of claim 29,wherein said polypeptide comprises an amino acid sequence having atleast about 95% identity with an amino acid sequence set forth in anyone of SEQ ID NOs: 13-24.
 36. The isolated polypeptide of claim 35,wherein said polypeptide comprises an amino acid sequence having atleast about 98% identity with an amino acid sequence set forth in anyone of SEQ ID NOs: 13-24.
 37. The isolated polypeptide of claim 29,wherein said polypeptide comprises an amino acid sequence set forth inany one of SEQ ID NOs: 13-24.
 38. The isolated polypeptide of claim 29,wherein said polypeptide is a mammalian polypeptide.
 39. The isolatedpolypeptide of claim 38, wherein said polypeptide is a humanpolypeptide.
 40. A kit for detecting a p63 protein comprising (i) anisolated anti-p63 antibody, or fragment thereof, and (ii) a means fordetecting the anti-p63 antibody.
 41. The kit of claim 40, wherein themeans for detecting the anti-p63 antibody is one or more of a buffer,formaldehyde, an enzyme, a co-enzyme, a substrate, a polypeptide, anantibody, or a detectable label.
 42. The kit of claim 41, wherein themeans for detecting the anti-p63 antibody is a detectable labelconjugated to the anti-p63 antibody.
 43. The kit of claim 41, whereinmeans for detecting the anti-p63 antibody is a second antibodyimmunoreactive with the anti-p63 antibody.
 44. The kit of claim 40,wherein the anti-p63 antibody binds to a p63 protein and does notdetectably bind to a p53 or p73 protein.
 45. The kit of claim 44,wherein the anti-p63 antibody is specifically immunoreactive with a p63protein.
 46. The kit of claim 40, wherein the anti-p63 antibody binds toa p63 protein comprising an amino acid sequence having at least about90% identity with a sequence set forth in any one of SEQ ID NOs: 13-24.47. The kit of claim 40, wherein the anti-p63 antibody binds to a p63protein comprising an amino acid sequence having a sequence set forth inany one of SEQ ID NOs: 13-24.
 48. The kit of claim 40, wherein theanti-p63 antibody is a monoclonal antibody.
 49. The kit of claim 40,wherein the anti-p63 antibody is a purified preparation of polyclonalantibodies.
 50. The kit of claim 40, wherein the anti-p63 antibody isprovided in a form suitable for detecting a p63 protein in cells.
 51. Akit for immunostaining a sample of cells, comprising: one or moreantibodies, or fragments thereof, for selectively binding p63 protein inthe sample, relative to p53 and p73; and a means for selectivelydetecting the antibody bound to p63 protein in the sample of cells. 52.The kit of claim 51, further comprising one or more reagents for fixingsaid sample of cells before immunostaining.
 53. The kit of claim 51,wherein the sample of cells is a tissue section.
 54. The kit of claim51, wherein the means for selectively detecting the antibody is an agentthat binds to the antibody and which includes a label that is detectableby spectrophotometry or fluorometry.
 55. The kit of claim 51, whereinthe means for selectively detecting the antibody is an agent that bindsto the antibody and which includes an enzyme that acts on a chromogenicsubstrate.
 56. The kit of claim 51, wherein the means for selectivelydetecting the antibody is a label that is detectable byspectrophotometry or fluorometry, or an enzyme that acts on achromogenic substrate, which label or enzyme is conjugated to theantibody.
 57. The kit of claim 51, wherein the antibody binds to a p63protein having an amino acid sequence set forth in any one of SEQ IDNOs: 13-24.
 58. The kit of claim 51, wherein the antibody is amonoclonal antibody.
 59. The kit of claim 51, wherein the one or moreantibodies is a preparation of p63-specific polyclonal antibodies. 60.The kit of claim 51, wherein the antibody differentially binds fordifferent isotypes of p63 proteins.
 61. The kit of claim 51, wherein theantibody binds to one or more p63 isotypes selected from the groupconsisting of TA*p63, TAp63, and ΔNp63.
 62. A kit for determining thelevel of p63 protein in a sample, comprising: an antibody, or fragmentthereof, which is selectively immunoreactive with a p63 protein,relative to p53 and p73 proteins; and a control protein including a p63peptide sequence that is bound by the antibody.